Pharmaceutical compositions for combination therapy

ABSTRACT

The present invention relates to a pharmaceutical composition comprising a combination of an FXR agonist and at least one lipid lowering agent (e.g., PPAR-alpha agonist, PPAR-delta agonist, PPAR-alpha and delta dual agonist, and/or statin). Also disclosed is use of the combination for the treatment or prevention of a FXR mediated disease or condition, such as primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), portal hypertension, bile acid diarrhea, NAFLD (nonalcoholic fatty liver disease), NASH (non-alcohol-induced steatohepatitis), and other chronic liver diseases. The combination of the present invention is useful for the treatment or prevention of conditions related to elevated lipid and liver enzyme levels. The present invention also relates to packs or kits including the pharmaceutical combination.

BACKGROUND TO THE INVENTION

Elevated concentrations of circulating lipid compounds in the blood,such as cholesterol and triglycerides, accompany a number of conditions.These include Type II diabetes, primary biliary cirrhosis (PBC), primarysclerosing cholangitis (PSC), various chronic hepatitis states(Hepatitis B and C), NASH (non-alcoholic steatohepatitis), and arterialdiseases including coronary artery disease, cerebrovascular arterialdisease, peripheral vascular disease, aortic aneurysms and carotidatherosclerotic conditions. Various lipid-lowering techniques have beenused in the past to treat and to prevent the vascular events (such ascardiac failure, embolism, heart attacks and strokes) that accompanyhyperlipidemic states. Such treatments have included dietary changes andcontrol of high triglyceride and cholesterol levels circulating in theblood. The latter have been treated generally pharmacologically andlately with various “statins”. Included in the therapeutic agents usedfor treatment of conditions for elevated lipid levels are various fibricacid derivatives. Some older fibric acid derivatives includingclofibrate have had a passing place in the treatment of conditionsassociated with elevated lipids, but more recently new fibratesincluding fenofibrate, gemfibrozil, ciprofibrate, and even more recentlyfibrates containing piperidine, 4-hydroxypiperidine, piperidin-3-ene,and piperazine have joined the ranks of anti-lipid therapies. Thesenewer molecules have promising properties to reduce both cholesterol andtriglycerides. However, in some situations a fibric acid derivativealone is inadequate in controlling the severe level of hyperlipidemiathat is present in many patients. The side effect profile of a fibricacid derivative may also be improved from a reduction in dose such as inthe presence of a combination therapy.

Accordingly, there is a need for an improved therapy for the treatmentof conditions involving elevated concentrations of circulating lipidcompounds in the blood, such as cholesterol and triglycerides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a representative photomicrograph of a Sirius-red stainedliver section from sham BDL mice.

FIG. 1B is a representative photomicrograph of a Sirius-red stainedliver section from BDL-vehicle treated mice.

FIG. 1C is a representative photomicrograph of Sirius-red stained liversection from BDL-OCA treated mice.

FIG. 1D is a representative photomicrograph of Sirius-red stained liversection from BDL-atorvastatin treated mice.

FIG. 1E is a representative photomicrograph of Sirius-red stained liversection from BDL-OCA-atorvastatin treated mice.

FIG. 2 is a bar graph showing the Sirius-red positive area (%) in BDLmice treated with OCA and atorvastatin alone and in combination.

FIG. 3A is a bar graph showing the number of inflammatory cell foci fromthe treatment of OCA, low dose fenofibrate alone and in combination inAPOE*3Leiden.CETP mice.

FIG. 3B is a bar graph showing the number of inflammatory cell foci fromthe treatment of OCA, high dose fenofibrate alone and in combination inAPOE*3Leiden.CETP mice.

FIG. 4 is a bar graph showing the effects of OCA and atorvastatin aloneand in combination on the fibrosis stage in leptin-ob/ob mice.

FIG. 5A is a bar graph showing the levels of plasma triglycerides inleptin-ob/ob mice treated with OCA and atorvastatin alone and incombination.

FIG. 5B is a bar graph showing the change in levels of plasmatriglycerides from baseline in leptin-ob/ob mice treated with OCA andatorvastatin alone and in combination.

FIG. 6A is a bar graph showing an enrichment analysis of the canonicalpathways of HFC+OCA against HFC sustained APOE*3Leiden.CETP mice.

FIG. 6B is a bar graph showing an enrichment analysis of the canonicalpathways of HFC+OCA+low dose fenofibrate against HFC sustainedAPOE*3Leiden.CETP mice

FIG. 7A is a venn diagram showing the number of novel differentiallyexpressed genes regulated by the combination of OCA+low dose fenofibrateversus monotherapy in APOE*3Leiden.CETP mice.

FIG. 7B is a bar graph showing the pathway enrichment of genes regulatedby the combination of OCA+low dose fenofibrate against monotherapy inAPOE*3Leiden.CETP mice.

FIG. 8 is a graph showing the effect of OCA and a combination of astatin(s) on LDL cholesterol in humans.

SUMMARY OF THE INVENTION

The present application relates to a pharmaceutical compositioncomprising (i) a first compound, (ii) at least one PPAR-alpha agonist,PPAR-delta agonist, and/or PPAR-alpha and delta dual agonist, and (iii)optionally one or more pharmaceutically acceptable carriers, wherein thefirst compound is an FXR agonist.

The present invention also relates to a pharmaceutical compositioncomprising (i) a first compound, (ii) at least one fibrate, andoptionally (iii) one or more pharmaceutically acceptable carriers,wherein the first compound is an FXR agonist.

The present invention also relates to a pharmaceutical compositioncomprising (i) a first compound, (ii) at least one lipid lowering agent,and optionally (iii) one or more pharmaceutically acceptable carriers,wherein the first compound is an FXR agonist.

The present invention also relates to a pharmaceutical compositioncomprising (i) a first compound, (ii) at least one statin, andoptionally (iii) one or more pharmaceutically acceptable carriers,wherein the first compound is an FXR agonist.

The present invention also relates to a pharmaceutical compositioncomprising (i) a first compound, (ii) at least one PPAR-alpha agonist,PPAR-delta agonist, and/or PPAR-alpha and delta dual agonist, (iii) atleast one lipid lowering agent, and optionally (iv) one or morepharmaceutically acceptable carriers, wherein the first compound is anFXR agonist.

The present invention also relates to a pharmaceutical compositioncomprising (i) a first compound, (ii) at least one fibrate, (iii) atleast one lipid lowering agent, and optionally (iv) one or morepharmaceutically acceptable carriers, wherein the first compound is anFXR agonist.

The present invention also relates to a pharmaceutical compositioncomprising (i) a first compound, (ii) at least one PPAR-alpha agonist,PPAR-delta agonist, and/or PPAR-alpha and delta dual agonist, (iii) atleast one statin, and optionally (iv) one or more pharmaceuticallyacceptable carriers, wherein the first compound is an FXR agonist.

The present invention also relates to a pharmaceutical compositioncomprising (i) a first compound, (ii) at least one fibrate, (iii) atleast one statin, and optionally (iv) one or more pharmaceuticallyacceptable carriers, wherein the first compound is an FXR agonist.

The present invention also relates to the therapeutic use of thepharmaceutical compositions of the present invention.

In one embodiment, the first compound is a compound of formula A:

or a pharmaceutically acceptable salt or amino acid conjugate thereof,wherein R₁, R₂, R₄, R₇, and X are as defined herein.

The present invention also relates to methods for treating or preventingan FXR mediated disease or condition or a disease or condition in whichelevated concentrations of circulating lipid compounds in the blood areinvolved, reducing the level of a liver enzyme, or inhibiting orreversing fibrosis, comprising administering a therapeutically effectiveamount of a pharmaceutical composition of the present invention to asubject in need thereof.

The present invention also relates to use of a pharmaceuticalcomposition of the present invention for treating or preventing an FXRmediated disease or condition or a disease or condition in whichelevated concentrations of circulating lipid compounds in the blood areinvolved, reducing the level of a liver enzyme, or inhibiting orreversing fibrosis.

The present invention also relates to use of a pharmaceuticalcomposition of the present invention in the manufacture of a medicamentfor treating or preventing an FXR mediated disease or condition or adisease or condition in which elevated concentrations of circulatinglipid compounds in the blood are involved, reducing the level of a liverenzyme, or inhibiting or reversing fibrosis.

The compositions and methods of the present invention address unmetneeds in the treatment or prevention of a disease or disorder in whichelevated concentrations of circulating lipid compounds in the blood,such as cholesterol and triglycerides, are involved.

DETAILED DESCRIPTION OF THE INVENTION

The present application is directed to a pharmaceutical compositioncomprising a first compound, at least one PPAR-alpha agonist, PPAR-deltaagonist, and/or PPAR-alpha and delta or PPAR-alpha and gamma dualagonist, and optionally one or more pharmaceutically acceptablecarriers, wherein the first compound is an FXR agonist.

In one example, the pharmaceutical composition comprises at least onePPAR-alpha agonist. In one example, the pharmaceutical compositioncomprises at least one PPAR-delta agonist. In one example, thepharmaceutical composition comprises at least one PPAR-alpha and deltadual agonist. In one example, the pharmaceutical composition comprisesat least one PPAR-alpha and gamma dual agonist. In one example, thepharmaceutical composition comprises at least one PPAR-alpha agonist andat least one PPAR-delta agonist. In one example, the pharmaceuticalcomposition comprises at least one PPAR-alpha agonist and at least onePPAR-alpha and delta dual agonist. In one example, the pharmaceuticalcomposition comprises at least one PPAR-delta agonist and at least onePPAR-alpha and delta or PPAR-alpha and gamma dual agonist. In oneexample, the pharmaceutical composition comprises at least onePPAR-alpha agonist, at least one PPAR-delta agonist, and at least onePPAR-alpha and delta dual agonist. In one example, the PPAR-alphaagonist is a fibrate, such as the fibrates described herein. In oneexample, the PPAR-delta agonist is{4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}aceticacid (also known as GW501516, GW1516 and “Endurabol”),{2-methyl-4-[5-methyl-2-(4-trifluoromethyl-phenyl)-2H-[1,2,3]triazol-4-ylmethylsylfanyl]-phenoxy}-aceticacid, or[4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]-acetic acid, or a pharmaceutically acceptable salt thereof. Inone example, the PPAR-alpha and delta dual agonist is 2-[2,6dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxyl]-2-methylpropanoicacid (also known as GFT505). In one example, the PPAR-alpha and gammadual agonist is aleglitazar((2S)-2-methoxy-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]-7-benzothiophenyl]propanoicacid), muraglitazar(N-[(4-methoxyphenoxy)carbonyl]-N-{4-[2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy]benzyl}glycine),tesaglitazar((2S)-2-ethoxy-3-[4-[2-(4-methylsulfonyloxyphenyl)ethoxy]phenyl]propanoicacid), or saroglitazar((2S)-2-ethoxy-3-[4-(2-{2-methyl-5-[4-(methylsulfanyl)phenyl]-1H-pyrrol-1-yl}ethoxy)phenyl]propanoicacid), or a pharmaceutically acceptable salt thereof. In one example,the PPAR-alpha and delta dual agonist is 2-[2,6dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxyl]-2-methylpropanoicacid, or a pharmaceutically acceptable salt thereof.

The present application is also directed to a pharmaceutical compositioncomprising a first compound, at least one fibrate, and optionally one ormore pharmaceutically acceptable carriers, wherein the first compound isan FXR agonist. The FXR agonist can be any FXR agonist. The fibrate canbe any fibrate. In one example, the fibrate is selected from anyfibrates described herein.

The present application is also directed to a pharmaceutical compositioncomprising a first compound, at least one lipid lowering agent, andoptionally one or more pharmaceutically acceptable carriers, wherein thefirst compound is an FXR agonist. The FXR agonist can be any FXRagonist. The lipid lowering agent can be any lipid lowering agent. Inone example, the lipid lowering agent is selected from any lipidlowering agents described herein.

The present application is also directed to a pharmaceutical compositioncomprising a first compound, at least one statin, and optionally one ormore pharmaceutically acceptable carriers, wherein the first compound isan FXR agonist. The FXR agonist can be any FXR agonist. The statin canbe any statin. In one example, the statin is selected from any statinsdescribed herein.

The present application is also directed to a pharmaceutical compositioncomprising a first compound, at least one PPAR-alpha agonist, PPAR-deltaagonist, and/or PPAR-alpha and delta or PPAR-alpha and gamma dualagonist, at least one lipid lowering agent, and optionally one or morepharmaceutically acceptable carriers, wherein the first compound is anFXR agonist.

The present application is also directed to a pharmaceutical compositioncomprising a first compound, at least one PPAR-alpha agonist, PPAR-deltaagonist, and/or PPAR-alpha and delta or PPAR-alpha and gamma dualagonist, at least one statin, and optionally one or morepharmaceutically acceptable carriers, wherein the first compound is anFXR agonist. In one example, the PPAR-alpha agonist is a fibrate, suchas the fibrates described herein. In one example, the PPAR-delta agonistis{4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}aceticacid,{2-methyl-4-[5-methyl-2-(4-trifluoromethyl-phenyl)-2H-[1,2,3]triazol-4-ylmethylsylfanyl]-phenoxy}-aceticacid, or[4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]-acetic acid, or a pharmaceutically acceptable salt thereof. Inone example, the PPAR-alpha and delta dual agonist is 2-[2,6dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxyl]-2-methylpropanoicacid. In one example, the PPAR-alpha and gamma dual agonist isaleglitazar, muraglitazar, tesaglitazar, or saroglitazar, or apharmaceutically acceptable salt thereof. In one example, the PPAR-alphaand delta dual agonist is 2-[2,6dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxyl]-2-methylpropanoicacid, or a pharmaceutically acceptable salt thereof. In one example, thelipid lowering agent is selected from any lipid lowering agentsdescribed herein. In one example, the statin is selected from anystatins described herein.

In one example, the first compound of the pharmaceutical composition isa compound of formula A:

or a pharmaceutically acceptable salt or amino acid conjugate thereof,wherein:

R₁ is hydrogen or unsubstituted C₁-C₆ alkyl;

R₂ is hydrogen or α-hydroxyl;

X is C(O)OH, C(O)NH(CH₂)_(m)SO₃H, C(O)NH(CH₂)_(n)CO₂H or OSO₃H;

R₄ is hydroxyl or hydrogen;

R₇ is hydroxyl or hydrogen;

m is 1, 2, or 3; and

n is 1, 2, or 3.

In a further example, the first compound of the pharmaceuticalcomposition is selected from formulae I and IA:

or a pharmaceutically acceptable salt or amino acid conjugate thereof,wherein

R_(1A) is hydrogen or unsubstituted C₁-C₆ alkyl;

R₂ is hydrogen or α-hydroxyl;

R₄ is hydroxyl or hydrogen; and

R₇ is hydroxyl or hydrogen.

In one aspect, the first compound is a pharmaceutically acceptable salt.In one embodiment, the first compound is a sodium salt of formula I orIA. In another embodiment, the first compound is an ammonium salt of acompound of formula I or IA. In another embodiment, the first compoundis a triethylammonium salt of a compound of formula I or IA.

In yet another example, the first compound of the pharmaceuticalcomposition is selected from formulae II and IIA:

or a pharmaceutically acceptable salt or amino acid conjugate thereof,wherein:

R_(1A) is hydrogen or unsubstituted C₁-C₆ alkyl;

R₂ is hydrogen or α-hydroxyl;

R₃ is hydroxyl, NH(CH₂)_(m)SO₃H, or NH(CH₂)_(n)CO₂H;

R₄ is hydroxyl or hydrogen;

R₇ is hydroxyl or hydrogen;

m is 1, 2, or 3; and

n is 1, 2, or 3.

In one example, the composition includes a first compound of formula A,I, IA, II or IIA, wherein R₂ is hydrogen.

In a further example, the composition includes a first compound offormula A, wherein R₁ is unsubstituted C₁-C₆ alkyl. In one aspect, thecomposition includes a first compound of formula A, wherein R₁ isunsubstituted C₁-C₃ alkyl. In one aspect, the composition includes afirst compound of formula A, wherein R₁ is selected from methyl, ethyl,and propyl. In one aspect, the composition includes a first compound offormula A, wherein R₁ is ethyl.

In a further example, the composition includes a first compound offormula I, IA, II, or IIA, wherein R_(1A) is unsubstituted C₁-C₆ alkyl.In one aspect, the composition includes a first compound of formula I,IA, II, or IIA, wherein R_(1A) is unsubstituted C₁-C₃ alkyl. In oneaspect, the composition includes a first compound of formula I, IA, II,or IIA, wherein R_(1A) is selected from methyl, ethyl, and propyl. Inone aspect, the composition includes a first compound of formula I, IA,II, or IIA, wherein R_(1A) is ethyl.

In a further example, the composition includes a first compound offormula A, wherein X is selected from C(O)OH, C(O)NH(CH₂)_(m)SO₃H, andC(O)NH(CH₂)_(n)CO₂H. In one aspect, the composition includes a firstcompound of formula A, wherein X is selected from C(O)OH,C(O)NH(CH₂)SO₃H, C(O)NH(CH₂)CO₂H, C(O)NH(CH₂)₂SO₃H, C(O)NH(CH₂)₂CO₂H. Inone aspect, the composition includes a first compound of formula A,wherein X is C(O)OH. In one aspect, the composition includes a firstcompound of formula A, wherein X is OSO₃H. In one aspect, thecomposition includes a first compound of formula A, wherein the firstcompound is a pharmaceutically acceptable salt. The pharmaceuticallyacceptable salt can be any salt. In one aspect, the composition includesa first compound of formula A, wherein X is OSO₃ ⁻Na⁺. In one aspect,the composition includes a first compound of formula A, wherein X isOSO₃ ⁻NHEt₃ ⁺. In one aspect, the amino acid conjugate is a glycineconjugate. In one aspect, the amino acid conjugate is a taurineconjugate.

In yet another example, the composition includes a first compound offormula II or IIA, wherein R₃ is selected from OH, NH(CH₂)SO₃H,NH(CH₂)CO₂H, NH(CH₂)₂SO₃H, and NH(CH₂)₂CO₂H. In one aspect, thecomposition includes a first compound of formula II or IIA, wherein R₃is OH.

In a further example, the composition includes a first compound offormula A, I, or II, wherein R₄ is hydroxyl and R₇ is hydrogen.

In a further example, the composition includes a first compound offormula A, wherein R₁ is selected from methyl, ethyl and propyl, R₄ isOH, R₇ is H, and R₂ is H.

In a further example, the composition includes a first compound offormula I or II, wherein R_(1A) is selected from methyl, ethyl andpropyl, R₄ is OH, R₇ is H, and R₂ is H.

In a further example, the composition includes a first compound offormula IA or IIA, wherein R_(1A) is selected from methyl, ethyl andpropyl, and R₂ is H.

In a further example, the composition includes a first compound selectedfrom

or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In yet a further example, the composition includes a first compound is apharmaceutically acceptable salt selected from

Compounds of formulae I, IA, II, and IIA are subsets of compounds offormula A. Features described herein for compounds of formula A applyequally to compounds of formulae I, IA, II, and IIA. The presentapplication also describes the pharmaceutical compositions, packs orkits, and therapeutic uses of the combination.

One of the problems to be solved by the present invention is theidentification of combination therapies for the treatment or preventionof conditions related to elevated concentrations of circulating lipidcompounds in the blood, such as cholesterol and triglycerides e.g., acholestatic liver condition such as PBC, as well as for the reduction ofcirculating lipid compounds (e.g., cholesterol, LDL, and triglycerides)in the blood, and for the reduction of bilirubin and/or liver enzymes,such as alkaline phosphatase (ALP, AP, or Alk Phos), alanineaminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltranspeptidase (GGT), lactate dehydrogenase (LDH), and 5′ nucleotidase.Although drugs for conditions related to elevated lipid levels and/orliver enzyme levels are available, these drugs are often not suitablefor many patients for a variety of reasons. For example, certain drugsare ineffective for patients who have developed drug resistance to,e.g., ursodeoxycholic acid. As another example, many statin drugs haveadverse effects such as muscle problems, cognitive loss, neuropathy,pancreatic and hepatic dysfunction, and sexual dysfunction. Some drugsmay be inadequate for the treatment when administered alone. Forexample, in some situations one lipid lowering agent alone is inadequatein controlling the severe level of hyperlipidemia that is present inmany patients. Some drugs may require administration of high doses, ormore frequent administration, due to extensive metabolism into inactiveor less potent metabolites. The combination therapies described hereincan solve the problems mentioned above and can have one or moreadvantages of, e.g., synergism, reducing the number of daily doseswithout the drug losing efficacy, lowering lipids (both cholesterol andtriglycerides) in patients whose elevated lipid levels are resistant totherapy in PBC, improved potency, selectivity, tissue penetration,half-life, and/or metabolic stability.

In the compositions, packs or kits, methods and uses of the presentinvention, the first compound may be the free acid or it may be apharmaceutically acceptable salt amino acid conjugate (e.g., glycine ortaurine conjugate). In one aspect, the first compound is any FXRagonist. In one aspect, the first compound is a compound of formula A.In one aspect, the first compound is a compound of formula I or IA. Inone aspect, the first compound is a compound of formula IA. In oneaspect, the first compound is a compound of formula II or IIA. In oneaspect, the first compound is a compound of formula IIA. In one aspect,the first compound is obeticholic acid (Compound 1). In one aspect, thefirst compound is Compound 2. In one aspect, the first compound is thepharmaceutically acceptable salt Compound 3. In one aspect, the firstcompound is the pharmaceutically acceptable salt Compound 4.

In the compositions, packs or kits, methods and uses of the presentinvention, the fibrate can be any fibrate. In one aspect, the fibrate isselected from the group consisting of fenofibrate, bezafibrate,beclobrate, binifibrate, ciprofibrate, clinofibrate, clofibrate,clofibric acid, etofibrate, gemfibrozil, nicofibrate, pirifibrate,ronifibrate, simfibrate, theofibrate, tocofibrate, plafibride, and apharmaceutically acceptable salt and ester thereof, and derivatives of2-phenoxy-2-methylpropanoic acid in which the phenoxy moiety issubstituted with an optionally substituted residue of piperidine,4-hydroxypiperidine, piperid-3-ene or piperazine, as disclosed inEuropean Patent Application Publication No. EP0607536. In one aspect,the fibrate is selected from the group consisting of bezafibrate,ciprofibrate, clofibrate, fenofibrate, gemfibrozil, binifibrate,clinofibrate, clofibric acid, nicofibrate, pirifibrate, plafibride,ronifibrate, theofibrate, tocofibrate, and a pharmaceutically acceptablesalt and ester thereof, and derivatives of 2-phenoxy-2-methylpropanoicacid, in which the phenoxy moiety is substituted with an optionallysubstituted residue of piperidine, 4-hydroxypiperidine, piperid-3-ene orpiperazine, as disclosed in European Patent Application Publication No.EP0607536. An example of the latter group of substances is2-[3-[1-(4-fluorobenzoyl)piperidin-4-yl]phenoxy-2-methyl-propanoic acid.For example, the fibrate is bezafibrate, fenofibrate, gemfibrozil,ciprofibrate, clofibrate, clofibric acid, or a pharmaceuticallyacceptable salt or ester thereof. For example, the fibrate isfenofibrate or a pharmaceutically acceptable salt selected from choline,ethanolamine, diethanolamine, piperazine, calcium, and tromethamine. Forexample, the fibrate is clofibrate or a pharmaceutically acceptable saltor ester thereof, such as etofibrate or aluminum clofibrate. Forexample, the fibrate is bezafibrate. For example, the fibrate is aderivative of 2-phenoxy-2-methylpropanoic acid such as2-[3-[1-(4-fluorobenzoyl)-piperidin-4-yl]phenoxyl-2-methylpropanoicacid.

In one embodiment, the first compound is the free acid of a compound offormula A, and the at least one fibrate is selected from bezafibrate,fenofibrate, gemfibrozil, ciprofibrate, clofibrate, and apharmaceutically acceptable salt or ester thereof.

In one embodiment, the first compound is a pharmaceutically acceptablesalt of compound of formula A, and the at least one fibrate is selectedfrom bezafibrate, fenofibrate, gemfibrozil, ciprofibrate, clofibrate,and a pharmaceutically acceptable salt or ester thereof.

In one embodiment, the first compound is the glycine conjugate of acompound of formula A, and the at least one fibrate is selected frombezafibrate, fenofibrate, gemfibrozil, ciprofibrate, clofibrate, and apharmaceutically acceptable salt or ester thereof.

In one embodiment, the first compound is the taurine conjugate of acompound of formula A, and the at least one fibrate is selected frombezafibrate, fenofibrate, gemfibrozil, ciprofibrate, clofibrate, andpharmaceutically acceptable salts or esters thereof.

In one embodiment, the first compound is a compound of formula A or apharmaceutically acceptable salt or amino acid conjugate, and the atleast one fibrate is2-[3-[1-(4-fluorobenzoyl)-piperidin-4-yl]phenoxyl-2-methylpropanoicacid.

In one embodiment, the first compound is a compound of formula A or apharmaceutically acceptable salt or amino acid conjugate, and the atleast one PPAR-delta agonist is{4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}aceticacid,{2-methyl-4-[5-methyl-2-(4-trifluoromethyl-phenyl)-2H-[1,2,3]triazol-4-ylmethylsylfanyl]-phenoxy}-aceticacid, or[4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]-acetic acid, or a pharmaceutically acceptable salt thereof.

In one embodiment, the first compound is a compound of formula A or apharmaceutically acceptable salt or amino acid conjugate, and the atleast one PPAR-alpha and delta dual agonist is 2-[2,6dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxyl]-2-methylpropanoicacid. In one embodiment, the first compound is a compound of formula Aor a pharmaceutically acceptable salt or amino acid conjugate, and theat least one PPAR-alpha and gamma dual agonist is aleglitazar,muraglitazar, tesaglitazar, or saroglitazar, or a pharmaceuticallyacceptable salt thereof. In one example, the PPAR-alpha and delta dualagonist is 2-[2,6dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxyl]-2-methylpropanoicacid, or a pharmaceutically acceptable salt thereof.

In the compositions, packs or kits, methods and uses of the presentinvention, the statin can be any statin. In one aspect, the statin isselected from the group consisting of simvastatin, fluvastatin,pravastatin, rivastatin, mevastatin, atorvastatin, cerivastatin,lovastatin, pitavastatin, fluindostatin, velostatin, dalvastatin,rosuvastatin, dihydrocompactin, and compactin.

In one embodiment, the first compound is the free acid of a compound offormula A, and the at least one statin is selected from simvastatin,fluvastatin, pravastatin, rivastatin, mevastatin, atorvastatin,cerivastatin, lovastatin, pitavastatin, fluindostatin, velostatin,dalvastatin, rosuvastatin, dihydrocompactin, and compactin.

In one embodiment, the first compound is a pharmaceutically acceptablesalt of compound of formula A, and the at least one statin is selectedfrom simvastatin, fluvastatin, pravastatin, rivastatin, mevastatin,atorvastatin, cerivastatin, lovastatin, pitavastatin, fluindostatin,velostatin, dalvastatin, rosuvastatin, dihydrocompactin, and compactin.

In one embodiment, the first compound is the glycine conjugate of acompound of formula A, and the at least one statin is selected fromsimvastatin, fluvastatin, pravastatin, rivastatin, mevastatin,atorvastatin, cerivastatin, lovastatin, pitavastatin, fluindostatin,velostatin, dalvastatin, rosuvastatin, dihydrocompactin, and compactin.

In one embodiment, the first compound is the taurine conjugate of acompound of formula A, and the at least one statin is selected fromsimvastatin, fluvastatin, pravastatin, rivastatin, mevastatin,atorvastatin, cerivastatin, lovastatin, pitavastatin, fluindostatin,velostatin, dalvastatin, rosuvastatin, dihydrocompactin, and compactin.

The invention also comprehends an isotopically-labeled first compound ora pharmaceutically acceptable salt or amino acid conjugate thereof,which has a structure that is identical to that of the first compound ofthe present invention (e.g., a compound of formula A, I, IA, II, orIIA), but for the fact that one or more atoms are replaced by an atomhaving an atomic mass or mass number different from the atomic mass ormass number most commonly found in nature. Examples of isotopes that canbe incorporated into the first compound or a pharmaceutically acceptablesalt or amino acid conjugate thereof, include isotopes of hydrogen,carbon, nitrogen, fluorine, such as ³H, ¹¹C, ¹⁴C and ¹⁸F.

The first compound or a pharmaceutically acceptable salt or amino acidconjugate thereof that contain the aforementioned isotopes and/or otherisotopes of other atoms is within the scope of the present invention.Isotopically-labeled first compound or a pharmaceutically acceptablesalt or amino acid conjugate thereof, for example, a first compound intowhich a radioactive isotopes such as ³H and/or ¹⁴C are incorporated, isuseful in drug and/or substrate tissue distribution assays. Tritiated,i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes are used for their ease ofpreparation and detectability. Further, substitution with heavierisotopes such as deuterium, i.e., ²H, can afford certain therapeuticadvantages resulting from greater metabolic stability, for exampleincreased in vivo half-life or reduced dosage requirements and, hence,may be used in some circumstances. Isotopically labeled first compoundor a pharmaceutically acceptable salt or amino acid conjugate thereofcan generally be prepared by carrying out the procedures disclosed inthe Schemes and/or in the Examples of the invention, by substituting areadily available isotopically labeled reagent for a non-isotopicallylabeled reagent. In one embodiment, obeticholic acid, orpharmaceutically acceptable salts or amino acid conjugates thereof arenot isotopically labelled.

The present invention also provides a method for treating or preventinga disease or condition, comprising administering a therapeuticallyeffective amount of a pharmaceutical composition of the presentinvention to a subject in need thereof.

In one embodiment, the disease or condition is an FXR mediated diseaseor condition. Examples of the FXR mediated diseases or conditionsinclude, but not limited to, liver diseases (including cholestatic andnon-cholestatic liver diseases) such as primary biliary cirrhosis (PBC),primary sclerosing cholangitis (PSC), biliary atresia, portalhypertension, bile acid diarrhea, chronic liver disease, nonalcoholicfatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH),hepatitis C infection, alcoholic liver disease, liver damage due toprogressive fibrosis, and liver fibrosis. Examples of FXR mediateddiseases also include hyperlipidemia, high LDL-cholesterol, highHDL-cholesterol, high triglycerides, and cardiovascular disease.

NAFLD is a medical condition that is characterized by the buildup of fat(called fatty infiltration) in the liver. NAFLD is one of the mostcommon causes of chronic liver disease, and encompasses a spectrum ofconditions associated with lipid deposition in hepatocytes. It rangesfrom steatosis (simple fatty liver), to nonalcoholic steatohepatitis(NASH), to advanced fibrosis and cirrhosis. The disease is mostly silentand is often discovered through incidentally elevated liver enzymelevels. NAFLD is strongly associated with obesity and insulin resistanceand is currently considered by many as the hepatic component of themetabolic syndrome.

Nonalcoholic steatohepatitis (NASH) is a condition that causesinflammation and accumulation of fat and fibrous (scar) tissue in theliver. Liver enzyme levels in the blood may be more elevated than themild elevations seen with nonalcoholic fatty liver (NAFL). Althoughsimilar conditions can occur in people who abuse alcohol, NASH occurs inthose who drink little to no alcohol. NASH affects 2 to 5 percent ofAmericans, and is most frequently seen in people with one of more of thefollowing conditions: obesity, diabetes, hyperlipidemia, insulinresistance, uses of certain medications, and exposure to toxins. NASH isan increasingly common cause of chronic liver disease worldwide and isassociated with increased liver-related mortality and hepatocellularcarcinoma, even in the absence of cirrhosis. NASH progresses tocirrhosis in 15-20% of affected individuals and is now one of theleading indications for liver transplantation in the United States. Atpresent there are no approved therapies for NASH.

In one embodiment, the disease or condition is hyperlipidemia. In oneembodiment, the disease or condition is a cholestatic liver disease. Inone embodiment, the disease or condition is PBC. In another embodiment,the disease or condition is a cardiovascular disease. In anotherembodiment, the cardiovascular disease is atherosclerosis,hypercholesterolemia, or hypertriglyceridemia.

The present invention also provides a method for treating or preventingNAFLD or NASH. In one embodiment, the present invention provides amethod for treating or preventing NAFLD or NASH that is associated withhyperlipidemia. In one embodiment, the present invention provides amethod for treating or preventing NASH. In one embodiment, the presentinvention provides a method for treating or preventing NASH that isassociated with hyperlipidemia.

The present invention also provides a method for inhibiting or reversingfibrosis, comprising administering a therapeutically effective amount ofa pharmaceutical composition of the present invention to a subject inneed thereof. In one embodiment, the subject is not suffering from acholestatic condition. In another embodiment, the subject is sufferingfrom a cholestatic condition.

In one embodiment, the subject is not suffering from a cholestaticcondition associated with a disease or condition selected from the groupconsisting of primary liver and biliary cancer (including hepatocellularcarcinoma), colorectal cancer, metastatic cancer, sepsis, chronic totalparenteral nutrition, cystic fibrosis, and granulomatous liver disease.In embodiments, the fibrosis to be inhibited or reversed occurs in anorgan where FXR is expressed.

In one embodiment, a cholestatic condition is defined as having anabnormally elevated serum level of alkaline phosphatase, γ-glutamyltranspeptidase (GGT), and/or 5′ nucleotidase. In another embodiment, acholestatic condition is further defined as presenting with at least oneclinical symptom. In one embodiment, the symptom is itching (pruritus).In another embodiment, a cholestatic condition is selected from thegroup consisting of primary biliary cirrhosis (PBC), primary sclerosingcholangitis (PBS), drug-induced cholestasis, hereditary cholestasis,biliary atresia, and intrahepatic cholestasis of pregnancy.

In one embodiment, the fibrosis is selected from the group consisting ofliver fibrosis, kidney fibrosis, and intestinal fibrosis.

In one embodiment, the subject has liver fibrosis associated with adisease selected from the group consisting of hepatitis B; hepatitis C;parasitic liver diseases; post-transplant bacterial, viral and fungalinfections; alcoholic liver disease (ALD); non-alcoholic fatty liverdisease (NAFLD); non-alcoholic steatohepatitis (NASH); liver diseasesinduced by methotrexate, isoniazid, oxyphenistatin, methyldopa,chlorpromazine, tolbutamide, or amiodarone; autoimmune hepatitis;sarcoidosis; Wilson's disease; hemochromatosis; Gaucher's disease; typesIII, IV, VI, IX and X glycogen storage diseases; α₁-antitrypsindeficiency; Zellweger syndrome; tyrosinemia; fructosemia; galactosemia;vascular derangement associated with Budd-Chiari syndrome,veno-occlusive disease, or portal vein thrombosis; and congenitalhepatic fibrosis.

In another embodiment, the subject has intestinal fibrosis associatedwith a disease selected from the group consisting of Crohn's disease,ulcerative colitis, post-radiation colitis, and microscopic colitis.

In another embodiment, the subject has renal fibrosis associated with adisease selected from the group consisting of diabetic nephropathy,hypertensive nephrosclerosis, chronic glomerulonephritis, chronictransplant glomerulopathy, chronic interstitial nephritis, andpolycystic kidney disease.

The present invention also provides a method for treating or preventingall forms of conditions related to elevated lipid levels. In oneembodiment, the condition is hyperlipidemia where it is associated witha condition selected from resistant primary biliary cirrhosis; primarybiliary cirrhosis where there is associated liver function testelevation and hyperlipidemia, primary sclerosing cholangitis,non-alcohol-induced steatohepatitis; and chronic liver diseaseassociated with hepatitis B, C or alcohol. In another embodiment, thepresent invention provides a method for treating or preventinghyperlipidemia where the hyperlipidemia is primary hyperlipidemia withor without a genetic component, or hyperlipidemia associated withcoronary artery disease, cerebrovascular arterial disease, peripheralvascular disease, aortic aneurisms, or carotid atherosclerosis.

In one aspect, the present invention provides a method for treating orpreventing primary sclerosing cholangitis for similar biochemicalabnormalities, as well as chronic hepatitis caused by hepatitis B, C orby alcohol. In one aspect, the present invention provides a method fortreating or preventing other arterial disorders associated withhyperlipidemia. In one aspect, the present invention provides a methodfor treating or preventing hypertriglyceridemia.

The present invention also provides a method for reducing lipid levels(i.e., amount of lipid), such as in the blood, comprising administeringa therapeutically effective amount of a pharmaceutical composition ofthe present invention to a subject in need thereof. In one embodiment,the method of the present invention reduces the lipid levels by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, as compared to a controlsubject (e.g., a subject not administered with the composition of thepresent invention). In one embodiment, the subject has elevated levelsof lipid, as compared to a healthy subject (e.g., an individual withouta disease or condition, such as those described herein). In oneembodiment, the method of the present application reduces the levels oflipid to normal levels (e.g., similar to the lipid levels in anindividual without a disease or condition, such as those describedherein).

In one embodiment, the lipid is cholesterol. In one embodiment, themethod of the present invention reduces cholesterol levels by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, as compared to a controlsubject (e.g., a subject not administered with the composition of thepresent invention). In one embodiment, the subject has elevated levelsof cholesterol, as compared to a healthy subject (e.g., an individualwithout a disease or condition, such as those described herein). In oneembodiment, the method of the present invention reduces cholesterollevels below 400 mg/L, 350 mg/L, 300 mg/L, 250 mg/L, 240 mg/L, 230 mg/L,220 mg/L, 210 mg/L, 200 mg/L, 190 mg/L, 180 mg/L, 170 mg/L, 160 mg/L, or150 mg/L. In one embodiment, the method of the present invention reducescholesterol levels below 200 mg/L, 190 mg/L, 180 mg/L, 170 mg/L, 160mg/L, or 150 mg/L.

In one embodiment, the cholesterol is LDL. In one embodiment, the methodof the present invention reduces LDL levels by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90%, as compared to a control subject (e.g.,a subject not administered with the composition of the presentinvention). In one embodiment, the subject has elevated levels of LDL,as compared to a healthy subject (e.g., an individual without a diseaseor condition, such as those described herein). In one embodiment, themethod of the present invention reduces LDL levels below 300 mg/L, 200mg/L, 190 mg/L, 180 mg/L, 170 mg/L, 160 mg/L, 150 mg/L, 140 mg/L, 130mg/L, 120 mg/L, 110 mg/L, 100 mg/L, 90 mg/L, 80 mg/L, 70 mg/L, 60 mg/L,or 50 mg/L.

In one embodiment, the method of the present invention reduces LDLlevels below 160 mg/L, 150 mg/L, 140 mg/L, 130 mg/L, 120 mg/L, 110 mg/L,100 mg/L, 90 mg/L, 80 mg/L, 70 mg/L, 60 mg/L, or 50 mg/L. In oneembodiment, the method of the present invention reduces LDL levels below130 mg/L, 120 mg/L, 110 mg/L, 100 mg/L, 90 mg/L, 80 mg/L, 70 mg/L, 60mg/L, or 50 mg/L. In one embodiment, the method of the present inventionreduces LDL levels below 100 mg/L, 90 mg/L, 80 mg/L, 70 mg/L, 60 mg/L,or 50 mg/L. In one embodiment, the method of the present inventionreduces LDL levels below 70 mg/L, 60 mg/L, or 50 mg/L.

In one embodiment, the lipid is triglyceride. In one embodiment, themethod of the present invention reduces triglyceride levels by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, as compared to a controlsubject (e.g., a subject not administered with the composition of thepresent invention). In one embodiment, the subject has elevated levelsof triglyceride, as compared to a healthy subject (e.g., an individualwithout a disease or condition, such as those described herein). In oneembodiment, the method of the present invention reduces triglyceridelevels below 800 mg/L, 700 mg/L, 600 mg/L, 500 mg/L, 400 mg/L, 300 mg/L,200 mg/L, 190 mg/L, 180 mg/L, 170 mg/L, 160 mg/L, 150 mg/L, 140 mg/L,130 mg/L, 120 mg/L, 110 mg/L, or 100 mg/L. In one embodiment, the methodof the present invention reduces triglyceride levels below 200 mg/L, 190mg/L, 180 mg/L, 170 mg/L, 160 mg/L, 150 mg/L, 140 mg/L, 130 mg/L, 120mg/L, 110 mg/L, or 100 mg/L. In one embodiment, the method of thepresent invention reduces triglyceride levels below 150 mg/L, 140 mg/L,130 mg/L, 120 mg/L, 110 mg/L, or 100 mg/L.

The present invention also provides a method for reducing the amount ofbilirubin, and/or one or more liver enzymes, comprising administering atherapeutically effective amount of a pharmaceutical composition of thepresent invention to a subject in need thereof.

In one embodiment, the method of the present application reduces theamount of bilirubin by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,or 90%, as compared to a control subject (e.g., a subject notadministered with the composition of the present invention). In oneembodiment, the subject has an elevated level of bilirubin, as comparedto a healthy subject (e.g., an individual without a disease orcondition, such as those described herein). In one embodiment, themethod of the present application reduces the level of bilirubin to anormal level (e.g., similar to the level of bilirubin in an individualwithout a disease or condition, such as those described herein). In afurther embodiment, the method of the present application reduces thelevel of bilirubin below 10 mg/L, 9 mg/L, 8 mg/L, 7 mg/L, 6 mg/L, 5mg/L, 4 mg/L, 3 mg/L, 2 mg/L, 1.5 mg/L, 1.2 mg/L, or 1 mg/L. In afurther embodiment, the method of the present application reduces thelevel of bilirubin below 2 mg/L, 1.5 mg/L, 1.2 mg/L, or 1 mg/L.

In one embodiment, the liver enzyme is selected from the groupconsisting of alkaline phosphatase (ALP, AP, or Alk Phos), alanineaminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltranspeptidase (GGT), lactate dehydrogenase (LDH), and 5′ nucleotidase.In one embodiment, the method of the present application reduces theamount of one or more liver enzymes by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90%, as compared to a control subject (e.g., a subjectnot administered with the composition of the present invention). In oneembodiment, the subject has elevated levels of one or more liverenzymes, as compared to a healthy subject (e.g., an individual without adisease or condition, such as those described herein). In oneembodiment, the method of the present application reduces the levels ofone or more liver enzymes (e.g., ALP, ALT, AST, GGT, LDH, and 5′nucleotidase) to normal levels (e.g., similar to the levels of liverenzymes in an individual without a disease or condition, such as thosedescribed herein).

In a further embodiment, the method of the present application reducesthe level of ALP below 500 IU/L (international units per liter), 400IU/L, 300 IU/L, 200 IU/L, 180 IU/L, 160 IU/L, or 150 IU/L. In a furtherembodiment, the method of the present application reduces the level ofALP to from about 40 IU/L to about 150 IU/L.

In a further embodiment, the method of the present application reducesthe level of ALT below 200 IU/L (international units per liter), 150IU/L, 100 IU/L, 80 IU/L, 60 IU/L, or 50 IU/L. In a further embodiment,the method of the present application reduces the level of ALT to fromabout 5 IU/L to about 50 IU/L.

In a further embodiment, the method of the present application reducesthe level of AST below 200 IU/L (international units per liter), 150IU/L, 100 IU/L, 80 IU/L, 60 IU/L, 50 IU/L, or 40 IU/L. In a furtherembodiment, the method of the present application reduces the level ofAST to from about 10 IU/L to about 50 IU/L.

In a further embodiment, the method of the present application reducesthe level of GGT below 200 IU/L (international units per liter), 150IU/L, 100 IU/L, 90 IU/L, 80 IU/L, 70 IU/L, or 60 IU/L. In a furtherembodiment, the method of the present application reduces the level ofGGT to from about 15 IU/L to about 50 IU/L or from about 5 IU/L to about30 IU/L.

In a further embodiment, the method of the present application reducesthe level of LDH below 500 IU/L (international units per liter), 400IU/L, 300 IU/L, 200 IU/L, 180 IU/L, 160 IU/L, 150 IU/L, 140 IU/L, or 130IU/L. In a further embodiment, the method of the present applicationreduces the level of LDH to from about 120 IU/L to about 220 IU/L.

In a further embodiment, the method of the present application reducesthe level of 5′ nucleotidase below 50 IU/L (international units perliter), 40 IU/L, 30 IU/L, 20 IU/L, 18 IU/L, 17 IU/L, 16 IU/L, 15 IU/L,14 IU/L, 13 IU/L, 12 IU/L, 11 IU/L, 10 IU/L, 9 IU/L, 8 IU/L, 7 IU/L, 6IU/L, or 5 IU/L. In a further embodiment, the method of the presentapplication reduces the level of 5′ nucleotidase to from about 2 IU/L toabout 15 IU/L.

In one embodiment, the methods of the present invention compriseadministering to a subject in need thereof an effective amount of afirst compound that is an FXR agonist, in combination with at least onePPAR-alpha agonist, PPAR-delta agonist, and/or PPAR-alpha and delta dualagonist, and optionally one or more pharmaceutically acceptablecarriers. In a further embodiment, the method comprises administering toa subject in need thereof an effective amount of a first compound, incombination with at least one PPAR-alpha agonist, PPAR-delta agonist,and/or PPAR-alpha and delta dual agonist, in which the first compound isa compound described herein (e.g., a compound of formula A, I, IA, II,or IIA, or Compound 1, 2, 3, or 4) or a pharmaceutically acceptable saltor amino acid conjugate thereof.

In one embodiment, the methods of the present invention compriseadministering to a subject in need thereof an effective amount of afirst compound that is an FXR agonist, in combination with at least onefibrate, and optionally one or more pharmaceutically acceptablecarriers. In a further embodiment, the method comprises administering toa subject in need thereof an effective amount of a first compound, incombination with at least one fibrate, in which the first compound is acompound described herein (e.g., a compound of formula A, I, IA, II, orIIA, or Compound 1, 2, 3, or 4) or a pharmaceutically acceptable salt oramino acid conjugate thereof.

In one embodiment, the methods of the present invention compriseadministering to a subject in need thereof an effective amount of afirst compound that is an FXR agonist, in combination with at least onestatin, and optionally one or more pharmaceutically acceptable carriers.In a further embodiment, the method comprises administering to a subjectin need thereof an effective amount of a first compound, in combinationwith at least one statin, in which the first compound is a compounddescribed herein (e.g., a compound of formula A, I, IA, II, or IIA, orCompound 1, 2, 3, or 4) or a pharmaceutically acceptable salt or aminoacid conjugate thereof.

In one embodiment, the methods of the present invention compriseadministering to a subject in need thereof an effective amount of afirst compound that is an FXR agonist, in combination with at least onePPAR-alpha agonist, PPAR-delta agonist, and/or PPAR-alpha and delta dualagonist, at least one statin, and optionally one or morepharmaceutically acceptable carriers. In a further embodiment, themethod comprises administering to a subject in need thereof an effectiveamount of a first compound, in combination with at least one PPAR-alphaagonist, PPAR-delta agonist, and/or PPAR-alpha and delta dual agonist,at least one statin, in which the first compound is a compound describedherein (e.g., a compound of formula A, I, IA, II, or IIA, or Compound 1,2, 3, or 4) or a pharmaceutically acceptable salt or amino acidconjugate thereof.

In one embodiment, the methods of the present invention compriseadministering to a subject in need thereof an effective amount of afirst compound that is an FXR agonist, in combination with at least onefibrate, at least one statin, and optionally one or morepharmaceutically acceptable carriers. In a further embodiment, themethod comprises administering to a subject in need thereof an effectiveamount of a first compound, in combination with at least one fibrate, atleast one statin, in which the first compound is a compound describedherein (e.g., a compound of formula A, I, IA, II, or IIA, or Compound 1,2, 3, or 4) or a pharmaceutically acceptable salt or amino acidconjugate thereof.

In one embodiment, the subject is a mammal. In one embodiment, themammal is human.

In one embodiment, the first compound and PPAR-alpha agonist(s),PPAR-delta agonist(s), PPAR-alpha and delta or PPAR-alpha and gamma dualagonist(s), fibrate(s), or statin(s) are administered in a two-waycombination, i.e., without any therapeutic agent other than the firstcompound and PPAR-alpha agonist(s), PPAR-delta agonist(s), PPAR-alphaand delta or PPAR-alpha and gamma dual agonist(s), fibrate(s), orstatin(s). In a further embodiment, the first compound and fibrate(s)are administered in a two-way combination, i.e., without any therapeuticagent other than the first compound and fibrate(s). In anotherembodiment, the first compound and statin(s) are administered in atwo-way combination, i.e., without any therapeutic agent other than thefirst compound and statin(s).

In another embodiment, the first compound and the PPAR-alpha agonist(s),PPAR-delta agonist(s), PPAR-alpha and delta or PPAR-alpha and gamma dualagonist(s), or fibrate(s) are administered in a three-way combinationwith a statin. In a further embodiment, the first compound andfibrate(s) are administered in a three-way combination with a statin.

The first compound, together with PPAR-alpha agonist(s), PPAR-deltaagonist(s), PPAR-alpha and delta or PPAR-alpha and gamma dualagonist(s), or fibrate(s), and/or statin(s) can achieve profoundsynergistic effects, such as synergistic reductions in severe, combinedhyperlipidemic states and those resistant to individual therapies and inthe levels of one or more liver enzymes. Hence, for the very difficultto control hyperlipidemias, a combination of the first compound, aPPAR-alpha agonist, a PPAR-delta agonist, a PPAR-alpha and delta orPPAR-alpha and gamma dual agonist, or a fibrate, and/or a statin isadvantageous. It can be particularly advantageous for such a combinationof the first compound, a fibrate, and/or a statin to be provided in asingle pharmaceutical composition with a pharmaceutical acceptablecarrier (such as in a single capsule form) designed to increasecompliance and hence effectiveness. Accordingly, the invention furtherprovides a pharmaceutical composition comprising an effective amount ofthe first compound, an effective amount of at least one PPAR-alphaagonist, PPAR-delta agonist, or PPAR-alpha and delta or PPAR-alpha andgamma dual agonist, and an effective amount of at least one statin,together with one or more pharmaceutically acceptable carriers,diluents, adjuvants or excipients. In one embodiment, the inventionfurther provides a pharmaceutical composition comprising an effectiveamount of the first compound, an effective amount of at least onefibrate, and an effective amount of at least one statin, together withone or more pharmaceutically acceptable carriers, diluents, adjuvants orexcipients.

In one embodiment, the first compound and PPAR-alpha agonist(s),PPAR-delta agonist(s), PPAR-alpha and delta or PPAR-alpha and gamma dualagonist(s), or fibrate(s) are administered concurrently. For example,the first compound and PPAR-alpha agonist(s), PPAR-delta agonist(s),PPAR-alpha and delta or PPAR-alpha and gamma dual agonist(s), orfibrate(s) are administered together in a single pharmaceuticalcomposition with a pharmaceutical acceptable carrier. In anotherembodiment, the first compound and PPAR-alpha agonist(s), PPAR-deltaagonist(s), PPAR-alpha and delta or PPAR-alpha and gamma dualagonist(s), or fibrate(s) are administered sequentially. For example,the first compound is administered prior or subsequent to PPAR-alphaagonist(s), PPAR-delta agonist(s), PPAR-alpha and delta or PPAR-alphaand gamma dual agonist(s), or fibrate(s).

In one embodiment, the first compound and the statin are administeredconcurrently. For example, the first compound and the statin areadministered together in a single pharmaceutical composition with apharmaceutical acceptable carrier. In another embodiment, the firstcompound and the statin are administered sequentially. For example, thefirst compound is administered prior or subsequent to the statin.

In one embodiment, the first compound is administered at a first dosefor a first time period, followed by administration of the firstcompound at a second dose for a second time period. In one embodiment, afirst compound or a pharmaceutically acceptable salt or amino acidconjugate thereof is administered in a daily total amount from 0.1-1500mg, 0.2-1200 mg, 0.3-1000 mg, 0.4-800 mg, 0.5-600 mg, 0.6-500 mg,0.7-400 mg, 0.8-300 mg, 1-200 mg, 1-100 mg, 1-50 mg, 1-30 mg, 4-26 mg,or 5-25 mg for a first time period, followed by administration of thefirst compound in a daily total amount from 0.1-1500 mg, 0.2-1200 mg,0.3-1000 mg, 0.4-800 mg, 0.5-600 mg, 0.6-500 mg, 0.7-400 mg, 0.8-300 mg,1-200 mg, 1-100 mg, 1-50 mg, 1-30 mg, 4-26 mg, or 5-25 mg. In oneembodiment, the total amount is orally administered once a day. In oneembodiment, the first dose is different from the second dose. In afurther embodiment, the first dose is lower than the second dose. Inanother embodiment, the first dose is higher than the second dose. Inone embodiment, the first dose is about 5 mg (e.g., from 4.8 mg to 5.2mg), and the second dose is about 10 mg (e.g., from 9.8 mg to 10.2 mg).In one embodiment, the first time period is about 6 months. In oneembodiment, the second time period is about 6 months.

In one embodiment, the pharmaceutical composition is administeredorally, parenterally, or topically. In another embodiment, thepharmaceutical composition is administered orally.

A composition in accordance with the present invention will typicallycontain sufficient first compound or a pharmaceutically acceptable saltor amino acid conjugate thereof, PPAR-alpha agonist(s), PPAR-deltaagonist(s), PPAR-alpha and delta or PPAR-alpha and gamma dualagonist(s), or fibrate(s), and/or statin(s) to permit the desired dailydose of each to be administered to a subject in need thereof in a singleunit dosage form, such as a tablet or capsule, or in two or more unitdosage forms to be administered simultaneously or at intervals during aday.

The invention also provides a pharmaceutical composition wherein thefirst compound and PPAR-alpha agonist(s), PPAR-delta agonist(s),PPAR-alpha and delta or PPAR-alpha and gamma dual agonist(s), orfibrate(s) are administered in combination with UDCA. In one aspect,UDCA is administered in a three-way combination. In another aspect, thetwo-way combination of a first compound and PPAR-alpha agonist(s),PPAR-delta agonist(s), PPAR-alpha and delta or PPAR-alpha and gamma dualagonist(s), or fibrate(s) is administered for the treatment orprevention of a disease or condition, in place of UDCA to a subject whohas an inadequate therapeutic response to UDCA alone.

In the methods of the present invention the active substances may beadministered in single daily doses, or in two, three, four or moreidentical or different divided doses per day, and they may beadministered simultaneously or at different times during the day.Usually, the active substances will be administered simultaneously, moreusually in a single combined dosage form.

In one aspect, the first compound, PPAR-alpha agonist(s), PPAR-deltaagonist(s), PPAR-alpha and delta or PPAR-alpha and gamma dualagonist(s), or fibrate(s), and/or statin(s) are administered at dosagessubstantially the same as the dosages at which they are administered inthe respective monotherapies. In one aspect, the first compound isadministered at a dosage which is less than (e.g., less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, or less than 10%) its monotherapy dosage.In one aspect, the PPAR-alpha agonist(s), PPAR-delta agonist(s),PPAR-alpha and delta or PPAR-alpha and gamma dual agonist(s), orfibrate(s) is administered at a dosage which is less than (e.g., lessthan 90%, less than 80%, less than 70%, less than 60%, less than 50%,less than 40%, less than 30%, less than 20%, or less than 10%) itsmonotherapy dosage. In one aspect, both the first compound andPPAR-alpha agonist(s), PPAR-delta agonist(s), PPAR-alpha and delta orPPAR-alpha and gamma dual agonist(s), or fibrate(s) are administered ata dosage which is less than (e.g., less than 90%, less than 80%, lessthan 70%, less than 60%, less than 50%, less than 40%, less than 30%,less than 20%, or less than 10%) their respective monotherapy dosages.In one aspect, the statin(s) is administered at a dosage which is lessthan (e.g., less than 90%, less than 80%, less than 70%, less than 60%,less than 50%, less than 40%, less than 30%, less than 20%, or less than10%) its monotherapy dosage. In one aspect, both the first compound andstatin(s) are administered at a dosage which is less than (e.g., lessthan 90%, less than 80%, less than 70%, less than 60%, less than 50%,less than 40%, less than 30%, less than 20%, or less than 10%) theirrespective monotherapy dosages. In one aspect, the first compound,PPAR-alpha agonist(s), PPAR-delta agonist(s), PPAR-alpha and delta orPPAR-alpha and gamma dual agonist(s), or fibrate(s), and/or statin(s)are administered at a dosage which is less than (e.g., less than 90%,less than 80%, less than 70%, less than 60%, less than 50%, less than40%, less than 30%, less than 20%, or less than 10%) their respectivemonotherapy dosages.

A pharmaceutical composition of the present invention may be in anyconvenient form for oral administration, such as a tablet, capsule,powder, lozenge, pill, troche, elixir, lyophilized powder, solution,granule, suspension, emulsion, syrup or tincture. Slow-release, modifiedrelease, or delayed-release forms may also be prepared, for example inthe form of coated particles, multi-layer tablets, capsules withincapsules, tablets within capsules, or microgranules.

Solid forms for oral administration may contain pharmaceuticallyacceptable binders, sweeteners, disintegrating agents, diluents,flavoring agents, coating agents, preservatives, lubricants and/or timedelay agents. Suitable binders include gum acacia, gelatin, corn starch,gum tragacanth, sodium alginate, carboxymethylellulose or polyethyleneglycol. Suitable sweeteners include sucrose, lactose, glucose, aspartameor saccharine. Suitable disintegrating agents include corn starch,methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginicacid or agar. Suitable diluents include lactose, sorbitol, mannitol,dextrose, kaolin, cellulose, calcium carbonate, calcium silicate ordicalcium phosphate. Suitable flavoring agents include peppermint oil,oil of wintergreen, cherry, orange or raspberry flavoring. Suitablecoating agents include polymers or copolymers or acrylic acid and/ormethacrylic acid and/or their esters, waxes, fatty alcohols, zein,shellac or gluten. Suitable preservatives include sodium benzoate,vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propylparaben or sodium bisulfite. Suitable lubricants include magnesiumstearate, stearic acid, sodium oleate, sodium chloride or talc. Suitabletime delay agents include glyceryl monostearate or glyceryl distearate.

Liquid forms for oral administration may contain, in addition to theabove agents, a liquid carrier. Suitable liquid carriers include water,oils such as olive oil, peanut oil, sesame oil, sunflower oil, saffloweroil, arachis oil, coconut oil, liquid paraffin, ethylene glycol,propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol,glycerol, fatty alcohols, triglycerides or mixtures thereof.

Suspensions for oral administration may further include dispersingagents and/or suspending agents. Suitable suspending agents includesodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, sodium alginate or cetyl alcohol.Suitable dispersing agents include lecithin, polyoxyethylene esters offatty acids such as stearic acid, polyoxyethylene sorbitol mono- ordi-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- ordi-oleate, -stearate or -laurate and the like.

Emulsions for oral administration may further include one or moreemulsifying agents. Suitable emulsifying agents include dispersingagents as exemplified above or natural gums such as gum acacia or gumtragacanth.

Pharmaceutical compositions of the present invention may be prepared byblending, grinding, homogenizing, suspending, dissolving, emulsifying,dispersing and/or mixing the first compound or its pharmaceuticallyacceptable salt or amino acid conjugate and at least one lipid loweringagent, e.g., fibrate, and optionally the statin(s) together with theselected excipient(s), carrier(s), adjuvant(s) and/or diluent(s). Onetype of pharmaceutical composition of the present invention in the formof a tablet or capsule may be prepared by (a) preparing a first tabletcomprising at least one of the active substances selected from the firstcompound or a pharmaceutically acceptable salt or amino acid conjugatethereof and at least one lipid lowering agent together with any desiredexcipient(s), carrier(s), adjuvant(s) and/or diluent(s), and (b)preparing a second tablet or a capsule, wherein the second tablet or thecapsule includes the remaining active substance(s) and the first tablet.Another type of pharmaceutical composition of the present invention inthe form of a capsule may be prepared by (a) preparing a first capsulecomprising at least one of the active substances selected from the firstcompound or a pharmaceutically acceptable salt or amino acid conjugatethereof and the lipid lowering agent(s), together with any desiredexcipient(s), carrier(s), adjuvant(s) and/or diluent(s), and (b)preparing a second capsule, wherein the second capsule includes theremaining active substance(s) and the first capsule. A further type ofpharmaceutical composition of the present invention in the form of atablet may be prepared by (a) preparing a capsule comprising at leastone of the active substances selected from a first compound or apharmaceutically acceptable salt or amino acid conjugate thereof and atleast one lipid lowering agent, together with any desired excipient(s),carrier(s), adjuvant(s) and/or diluent(s), and (b) preparing a tablet,wherein the tablet includes the remaining active substance(s) and thecapsule.

In embodiments, the PPAR-alpha agonist(s), PPAR-delta agonist(s),PPAR-alpha and delta or PPAR-alpha and gamma dual agonist(s), orfibrate(s), and/or statin(s) is used either as an immediate releasetablet or as a sustained release tablet. It is particularly effectivewhen provided in a sustained release tablet. Sustained release tabletsof various lipid lowering agents are commercially available. It ispreferable for prolonged action that the tablet is in a sustainedrelease format.

In another embodiment, the pharmaceutical composition of the presentinvention comprises a capsule containing a PPAR-alpha agonist(s),PPAR-delta agonist(s), PPAR-alpha and delta or PPAR-alpha and gamma dualagonist(s), or fibrate(s), and/or statin(s) within a capsule containinga first compound or a pharmaceutically acceptable salt or amino acidconjugate thereof. Typically in this form the PPAR-alpha agonist(s),PPAR-delta agonist(s), PPAR-alpha and delta or PPAR-alpha and gamma dualagonist(s), or fibrate(s), and/or statin(s) is presented in an immediaterelease form. In that event it is usual to administer the compositionthree times daily. Another mode of administration is to provide acomposition containing the PPAR-alpha agonist(s), PPAR-delta agonist(s),PPAR-alpha and delta or PPAR-alpha and gamma dual agonist(s), orfibrate(s), and/or statin(s) in either a sustained release or anon-sustained release form as described above, twice daily, wherein thedaily amount of the composition administered contains sufficient amountof the active substances to provide the desired daily dosage to thepatient.

In one embodiment, the pharmaceutical compositions of the invention is adosage form which comprises a first compound or a pharmaceuticallyacceptable salt or amino acid conjugate thereof in a daily total amountof from 0.1-1500 mg, 0.2-1200 mg, 0.3-1000 mg, 0.4-800 mg, 0.5-600 mg,0.6-500 mg, 0.7-400 mg, 0.8-300 mg, 1-200 mg, 1-100 mg, 1-50 mg, 1-30mg, 4-26 mg, or 5-25 mg. In one embodiment, the total amount is orallyadministered once a day.

In one embodiment, the pharmaceutical compositions of the invention is adosage form which comprises a PPAR-alpha agonist, PPAR-delta agonist,PPAR-alpha and delta or PPAR-alpha and gamma dual agonist, or fibrate,and/or statin in a daily total amount of 10-1000 mg, 20-800 mg, 50-500mg, 80-400 mg, or 100-300 mg, more typically about 200 mg. In oneembodiment, the total amount is orally administered once a day. In oneembodiment, the pharmaceutical compositions of the invention is a dosageform which comprises a statin in an amount of 5-1000 mg, 10-800 mg,20-500 mg, 30-400 mg, or 40-200 mg.

In embodiments, the compositions of the invention is a dosage form whichcomprises a PPAR-alpha agonist, PPAR-delta agonist, PPAR-alpha and deltaor PPAR-alpha and gamma dual agonist, or fibrate, and/or statin in anamount of 10-1000 mg, 20-800 mg, 50-500 mg, 80-400 mg, or 100-300 mg,more typically about 200 mg, contained within a capsule which containsthe first compound in an amount of from 0.1-1500 mg, 0.2-1200 mg,0.3-1000 mg, 0.4-800 mg, 0.5-600 mg, 0.6-500 mg, 0.7-400 mg, 0.8-300 mg,1-200 mg, 1-100 mg, 1-50 mg, 1-30 mg, 4-26 mg, or 5-25 mg. In oneembodiment, the PPAR-alpha agonist, PPAR-delta agonist, PPAR-alpha anddelta or PPAR-alpha and gamma dual agonist, or fibrate, and/or statin isin the sustained release form.

In embodiments, the compositions of the invention is a dosage form whichcomprises a sustained release tablet of bezafibrate, in an amount of10-1000 mg, 20-800 mg, 50-500 mg, 80-400 mg, or 100-300 mg, moretypically about 200 mg, contained within a capsule which contains thefirst compound in an amount of from 0.1-1500 mg, 0.2-1200 mg, 0.3-1000mg, 0.4-800 mg, 0.5-600 mg, 0.6-500 mg, 0.7-400 mg, 0.8-300 mg, 1-200mg, 1-100 mg, 1-50 mg, 1-30 mg, 4-26 mg, or 5-25 mg. In this way thepatient to whom the dosage form is administered receives a sustainedrelease tablet of bezafibrate which is delivered to the distal antrum asthe capsule breaks open and releases the first compound.

The pharmaceutical composition of the present invention can be usedlifelong by the patient, prolonging survival and delaying livertransplantation. The reduction of hyperlipidemia and liver enzymesensures reduction in the development of associated vascular disease.Both the first compound and lipid lowering agents, such as fibratesand/or statins have very minimal long-term side effect profile (withsome exceptions for bezafibrate) and therefore this combination islikely to be the therapy of choice for primary biliary cirrhosis (PBC)with hyperlipidemia and for resistant primary biliary cirrhosis (PBC).Because of the simplified dosing provided by the present invention, acombined therapy of the present invention can be used in increasingdoses, depending on a patient's weight and clinical response.

A composition of the present invention that comprises a first compoundor a pharmaceutically acceptable salt or amino acid conjugate thereof, aPPAR-alpha agonist, PPAR-delta agonist, PPAR-alpha and delta orPPAR-alpha and gamma dual agonist, or fibrate, and/or a statin can beprovided as the three active substances within a single capsule. In oneform of such a composition, a statin may be mixed with a first compoundin an inner capsule, the inner capsule being surrounded by a PPAR-alphaagonist, PPAR-delta agonist, PPAR-alpha and delta or PPAR-alpha andgamma dual agonist, or fibrate contained within an outer capsule. Thelocations within the capsules may be reversed. That is, the mixture of astatin and a first compound may be contained within the outer capsuleand the PPAR-alpha agonist, PPAR-delta agonist, PPAR-alpha and delta orPPAR-alpha and gamma dual agonist, or fibrate may be contained withinthe inner capsule. This arrangement will be especially desirable if thequantity of the statin to be administered is relatively large. Othercombinations for administration of the combination of three activesubstances are possible.

The first compounds disclosed herein can be prepared by the conventionalmethods (e.g., those described in U.S. Publication No. 2009/0062526,U.S. Pat. No. 7,138,390, and WO 2006/122977), such as by a 6-stepsynthesis followed by one purification step to produce highly pureCompound 1 (obeticholic acid, or OCA) as shown in Scheme 1 below.

The process above was described in WO 2013/192097, the contents of whichare incorporated herein by reference in their entirety. The process is a6-step synthesis followed by one purification step. Step 1 is theesterification of the C-24 carboxylic acid of 7-keto lithocholic acid(KLCA) to produce the methyl ester compound a. Step 2 is silylenol etherformation from compound 1 to produce compound c. Step 3 is an aldolcondensation reaction of the silylenol ether compound c and acetaldehydeto produce compound d. Step 4 is saponification of compound d to producecompound e. Step 5 is the hydrogenation of compound e to producecompound f. Step 6 is the selective reduction of the 7-keto group ofcompound f to produce crystalline Compound 1. Step 7 is the conversionof crystalline compound to amorphous Compound 1 (obeticholic acid Form1, or OCA Form 1).

Alternatively, the first compound disclosed herein can be prepared bythe conventional methods (e.g., those described in U.S. Pat. No.7,932,244), or via a process as shown in Scheme 2 (and disclosed WO2014/066819). Scheme 2 can be used to prepare Compound 2, 3, or 4disclosed herein.

Step 1 is the esterification of a compound of formula II to obtain acompound of formula III. Step 2 is a reaction to form a compound offormula IV from a compound of formula III. Step 3 is the protection ofthe hydroxy group at the C3 position of a compound of formula IV toafford a compound of formula V. Step 4 is the oxidative cleavage ofcompound of formula V to give a compound of formula VI. Step 5 is thereduction of a compound of formula VI to afford a compound of formulaVII. Step 6 is the sulfonation of a compound of formula VII to give asalt of formula I-Na. A salt of formula I—Na can be converted to itsfree base form (i.e., a compound of formula I) or other salt forms(e.g., a salt of formula I-(Et)₃NH).

Definitions

For convenience, certain terms used in the specification, examples andappended claims are collected here.

As used herein the term “fibrate” means any of fibric acid derivativesand pharmaceutically active derivatives of 2-phenoxy-2-methylpropanoicacid useful in the methods described herein. Examples of fibratesinclude, but are not limited to, fenofibrate, bezafibrate, beclobrate,binifibrate, ciprofibrate, clinofibrate, clofibrate, clofibric acid,etofibrate, gemfibrozil, nicofibrate, pirifibrate, ronifibrate,simfibrate, theofibrate, tocofibrate, plafibride, etc. Examples offibrates are also described in U.S. Pat. Nos. 3,781,328, 3,948,973,3,869,477, 3,716,583, 3,262,580, 3,723,446, 4,058,552, 3,674,836,3,369,025, 3,984,413, 3,971,798, 6,384,062, 7,119,198 and 7,259,186;U.S. Pub. No. 20090131395; WO2008/039829; Belgian patent no. 884722;United Kingdom patent no. 860303; and European patent applicationpublication no. EP0607536, the entire disclosures of each of which areincorporated herein by reference.

Peroxisome proliferator-activated receptor alpha (PPAR-alpha), alsoknown as NR1C1 (nuclear receptor subfamily 1, group C, member 1), is anuclear receptor protein. A PPAR-alpha agonist binds to and activatesPPAR-alpha. Examples of a PPAR-alpha agonist include, but are notlimited to, a fibrate, such as the fibrates described herein.

Peroxisome proliferator-activated receptor delta (PPAR-delta), alsoknown as NR1C2 (nuclear receptor subfamily 1, group C, member 2), is anuclear receptor protein. A PPAR-delta agonist binds to and activatesPPAR-delta. Examples of a PPAR-delta agonist include, but are notlimited to,{4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}aceticacid (also known in the art as GW501516, GW1516, and Endurabol),{2-methyl-4-[5-methyl-2-(4-trifluoromethyl-phenyl)-2H-[1,2,3]triazol-4-ylmethylsylfanyl]-phenoxy}-aceticacid, and[4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]-acetic acid.

A PPAR-alpha and delta or PPAR-alpha and gamma dual agonist binds to andactivates both PPAR-alpha and PPAR-delta, or both PPAR-alpha andPPAR-gamma. Examples of PPAR-alpha and delta dual agonist include, butare not limited to, 2-[2,6dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxyl]-2-methylpropanoicacid (also known as GFT505). Examples of PPAR_alpha and gamma dualagonists include, but are not limited to, aleglitazar((2S)-2-methoxy-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]-7-benzothiophenyl]propanoicacid, CAS No. 475479-34-6), muraglitazar(N-[(4-methoxyphenoxy)carbonyl]-N-{4-[2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy]benzyl}glycine,CAS No. 331741-94-7), tesaglitazar((2S)-2-ethoxy-3-[4-[2-(4-methylsulfonyloxyphenyl)ethoxy]phenyl]propanoicacid, CAS No. 251565-85-2) and saroglitazar((2S)-2-ethoxy-3-[4-(2-{2-methyl-5-[4-(methylsulfanyl)phenyl]-1H-pyrrol-1-yl}ethoxy)phenyl]propanoicacid, CAS No. 495399-09-2).

As used herein, the term “FXR agonist” refers to any compound whichactivates FXR. In one aspect, an FXR agonist achieves at least 50%activation of FXR relative to CDCA, the appropriate positive control inthe assay methods described in WO 2000/037077. In another aspect, an FXRagonist achieves 100% activation of FXR in the scintillation proximityassay or the HTRF assay as described in WO2000/037077. Examples of FXRagonists include but are not limited to those described in U.S. Pat.Nos. 7,138,390; 7,932,244; 20120283234; 20120232116;20120053163;20110105475;20100210660;20100184809;20100172870;20100152166;20100069367;20100063018;20100022498;20090270460;20090215748;20090163474;20090093524; 20080300235; 20080299118; 20080182832; 20080039435;20070142340; 20060069070; 20050080064; 20040176426; 20030130296;20030109467; 20030003520; 20020132223; and 20020120137.

As used herein, the term “obeticholic acid” or “OCA” refers to acompound having the chemical structure:

Obeticholic acid is also referred to as obeticholic acid Form 1,INT-747, 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid,6α-ethyl-chenodeoxycholic acid, 6-ethyl-CDCA, 6ECDCA, cholan-24-oicacid, 6-ethyl-3,7-dihydroxy-(3α,5β, 6α,7α), and can be prepared by themethods described in U.S. Publication No. 2009/0062526 A1, U.S. Pat. No.7,138,390, and WO2006/122977. The CAS registry number for obeticholicacid is 459789-99-2.

As used herein, the term “crystalline obeticholic acid” refers to anycrystalline form of a compound having the chemical structure:

Crystalline obeticholic acid means that the compound is crystallizedinto a specific crystal packing arrangement in three spatial dimensionsor the compound having external face planes. The crystalline form ofobeticholic acid (or a pharmaceutically acceptable salt thereof) cancrystallize into different crystal packing arrangements, all of whichhave the same elemental composition of obeticholic acid. Differentcrystal forms usually have different X-ray diffraction patterns,infrared spectral, melting points, density hardness, crystal shape,optical and electrical properties, stability and solubility.Recrystallization solvent, rate of crystallization, storage temperature,and other factors may cause one crystal form to dominate. Crystals ofobeticholic acid can be prepared by crystallization under differentconditions, e.g., different solvents, temperatures, etc. Examples ofcrystalline forms of OCA are described in U.S. Pat. No. 9,238,673.

The term “first compound” means a compound of formula A, I, IA, II, orIIA, or Compound 1, 2, 3, or 4, or a pharmaceutically acceptable salt oramino acid conjugate thereof. Whenever the term is used in the contextof the present invention it is to be understood that the reference isbeing made to the free base, an isotopically-labeled compound, acrystalline compound, or a corresponding pharmaceutically acceptablesalt or amino acid conjugates thereof, provided that such is possibleand/or appropriate under the circumstances.

As used herein, the term “amino acid conjugates” refers to conjugates ofa first compound of the present invention (e.g., a compound of FormulaA) with any suitable amino acid. For example, such a suitable amino acidconjugate of a compound of Formula A will have the added advantage ofenhanced integrity in bile or intestinal fluids. Suitable amino acidsinclude but are not limited to glycine and taurine. Thus, the presentinvention encompasses the glycine and taurine conjugates of a firstcompound of the present invention (e.g., Compound 1).

The term “statin” is synonymous with the terms“3-hydroxy-3-methylglutaryl-Coenzyme A reductase inhibitor” and “HMG-CoAreductase inhibitor”. These terms are used interchangeably herein. Asthe synonyms suggest, statins are inhibitors of3-hydroxy-3-methylglutaryl-Coenzyme A reductase and, as such, areeffective in lowering the level of blood plasma cholesterol andaccordingly for treating or preventing cardiovascular diseases. Statinsand pharmaceutically acceptable salts thereof are particularly useful inlowering low-density lipoprotein cholesterol (LDL-C) levels in mammalsand particularly in humans. Structurally, statins or derivatives thereofhave in common a 4-hydroxy-6-oxo-2H-pyran system, which may also be inthe form of dihydroxy acid which interacts with the active site ofHMG-CoA reductase, and a lipophilic part which presents in particular asa polysubstituted hexahydronaphthalenic system, but may also be replacedwith a polysubstituted heteroaromatic system, as in atorvastatin orfluvastatin. The statin suitable for use herein include, but are notlimited to, simvastatin, fluvastatin, pravastatin, rivastatin,mevastatin, atorvastatin, cerivastatin, lovastatin, pitavastatin,fluindostatin, velostatin, dalvastatin, rosuvastatin, dihydrocompactin,and compactin, or a pharmaceutically acceptable salt thereof.

The term “lipid lowering agent” refers to any agent that is capable oflowering the concentration of lipid (e.g., cholesterol, LDL, andtriglyceride) in circulation (e.g., in the blood). A lipid loweringagent includes, but is not limited to, (i) a bile acid sequestrant, suchas a resin (e.g., cholestyramine, colestipol, colesevelam), (ii) acholesterol absorption inhibitor, which prevents uptake of cholesterol(e.g., from the small intestine into the circulatory system), such asezetimibe (i.e.,(3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one)and (3R,4S)-1,4-bis(4-methoxyphenyl)-3-(3-phenylpropyl)-2-azetidinone,(iii) Omega-3 fatty acid ethyl esters, including free fatty acidderivatives (e.g., Omacor®, Lovaza®, Vascepa™, Epadel, Epanova™), ormarine-derived omega-3 polyunsaturated fatty acids (PUFA), (iv) PCSK9inhibitors, (v) nicotinic acid, (vi) phytosterols (e.g., plant sterolsand stanols), such as β-sitosterol, campesterol, stigmasterol,brassicasterol, ergosterol, β-sitostanol, campestanol, stigmastanol,cycloartenol, and lupeol, (vii) inhibitors of CETP (cholesteryl estertransfer protein), such as Anacetrapib, Evacetrapib, Torcetrapib, andDalcetrapib, (viii) squalene synthase inhibitors, (ix) antisenseoligonucleotides which affect the synthesis, degredation, absorption,and metabolism of lipids (e.g., antisense oligonucleotides that binds tothe mRNA that encodes apolipoprotein B or PCSK9) (e.g., Mipomersen(Kynamro)), (x) apoprotein-B inhibitors, (xi) inhibitors of microsomaltriglyceride transport protein (e.g., Lomitapide (Juxtapid)), and (xii)other compounds, such as colesevelam, avasimibe, and implitapide.

“Treating”, includes any effect, e.g., lessening, reducing, modulating,or eliminating, that results in the improvement of the condition,disease, disorder, etc. “Treating” or “treatment” of a disease stateincludes: inhibiting the disease state, i.e., arresting the developmentof the disease state or its clinical symptoms, or relieving the diseasestate, i.e., causing temporary or permanent regression of the diseasestate or its clinical symptoms.

“Preventing” the disease state includes causing the clinical symptoms ofthe disease state not to develop in a subject that may be exposed to orpredisposed to the disease state, but does not yet experience or displaysymptoms of the disease state.

The term “inhibiting” or “inhibition,” as used herein, refers to anydetectable positive effect on the development or progression of adisease or condition. Such a positive effect may include the delay orprevention of the onset of at least one symptom or sign of the diseaseor condition, alleviation or reversal of the symptom(s) or sign(s), andslowing or prevention of the further worsening of the symptom(s) orsign(s).

“Disease state” means any disease, disorder, condition, symptom, orindication.

The term “effective amount” or “therapeutically effective amount” asused herein refers to an amount of a first compound (e.g., anFXR-activating ligand), or a fibrate, or a lipid lowering agent, or astatin that produces an acute or chronic therapeutic effect uponappropriate dose administration, alone or in combination. In oneembodiment, an effective amount or therapeutically effective amount of afirst compound (e.g., an FXR-activating ligand) produces an acute orchronic therapeutic effect upon appropriate dose administration incombination with at least one fibrate. The effect includes theprevention, correction, inhibition, or reversal of the symptoms, signsand underlying pathology of a disease/condition (e.g., fibrosis of theliver, kidney, or intestine) and related complications to any detectableextent. An “effective amount” or “therapeutically effective amount” willvary depending on the first compound, the fibrate, the lipid loweringagent, the statin, the disease and its severity, and the age, weight,etc., of the subject to be treated.

A therapeutically effective amount of a first compound can be formulatedtogether with one or more fibrates, and optionally one or morepharmaceutically acceptable carriers for administration to a human or anon-human animal. Accordingly, the pharmaceutical composition of theinvention can be administered, for example, via oral, parenteral, ortopical routes, to provide an effective amount of the first compound andthe fibrate(s). In alternative embodiments, the compositions of theinvention can be used to coat or impregnate a medical device, e.g., astent.

“Pharmacological effect” as used herein encompasses effects produced inthe subject that achieve the intended purpose of a therapy. In oneembodiment, a pharmacological effect means that primary indications ofthe subject being treated are prevented, alleviated, or reduced. Forexample, a pharmacological effect would be one that results in theprevention, alleviation or reduction of primary indications in a treatedsubject. In another embodiment, a pharmacological effect means thatdisorders or symptoms of the primary indications of the subject beingtreated are prevented, alleviated, or reduced. For example, apharmacological effect would be one that results in the prevention,alleviation or reduction of the disorders or symptoms in a treatedsubject.

It is to be understood that the isomers arising from asymmetric carbonatoms (e.g., all enantiomers and diastereomers) are included within thescope of the invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis.

A “pharmaceutical composition” is a formulation containing therapeuticagents such as a first compound and a lipid lowering agent, such as afibrate, in a form suitable for administration to a subject. In oneembodiment, the pharmaceutical composition is in bulk or in unit dosageform. It can be advantageous to formulate compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active reagent calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active agents and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active agent for the treatment of individuals.

The term “unit dosage form” refers to physically discrete units suitableas unitary dosages for humans and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient as described herein.

The unit dosage form is any of a variety of forms, including, forexample, a capsule, an IV bag, a tablet, a single pump on an aerosolinhaler, or a vial. The quantity of first compound or a pharmaceuticallyacceptable salt or amino acid conjugate thereof in a unit dose ofcomposition is an effective amount and is varied according to theparticular treatment involved and/or the lipid lowering agent(s) usedfor the treatment. One skilled in the art will appreciate that it issometimes necessary to make routine variations to the dosage dependingon the age and condition of the patient. The dosage will also depend onthe route of administration. A variety of routes are contemplated,including oral, pulmonary, rectal, parenteral, transdermal,subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational,buccal, sublingual, intrapleural, intrathecal, intranasal, and the like.Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. In one embodiment, thefirst compound and/or a lipid lowering agent is mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that are required.

The term “flash dose” refers to formulations that are rapidly dispersingdosage forms.

The term “immediate release” is defined as a release of a therapeuticagent (such as a first compound or lipid lowering agent) from a dosageform in a relatively brief period of time, generally up to about 60minutes. The term “modified release” is defined to include delayedrelease, extended release, and pulsed release. The term “pulsed release”is defined as a series of releases of drug from a dosage form. The term“sustained release” or “extended release” is defined as continuousrelease of a therapeutic agent from a dosage form over a prolongedperiod.

A “subject” includes mammals, e.g., humans, companion animals (e.g.,dogs, cats, birds, and the like), farm animals (e.g., cows, sheep, pigs,horses, fowl, and the like), and laboratory animals (e.g., rats, mice,guinea pigs, birds, and the like). In one embodiment, the subject ishuman. In one aspect, the subject is female. In one aspect, the subjectis male.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable carrier or excipient” means a carrier orexcipient that is useful in preparing a pharmaceutical composition thatis generally safe, non-toxic and neither biologically nor otherwiseundesirable, and includes excipient that is acceptable for veterinaryuse as well as human pharmaceutical use. A “pharmaceutically acceptableexcipient” as used in the specification and claims includes both one andmore than one such excipient.

While it is possible to administer the first compound directly withoutany formulation, the first compound may be administered in the form of apharmaceutical formulation comprising a pharmaceutically acceptableexcipient. This formulation can be administered by a variety of routesincluding oral, buccal, rectal, intranasal, transdermal, subcutaneous,intravenous, intramuscular, and intranasal.

In one embodiment, the first compound can be administered transdermally.In order to administer transdermally, a transdermal delivery device(“patch”) is needed. Such transdermal patches may be used to providecontinuous or discontinuous infusion of a compound of the presentinvention in controlled amounts. The construction and use of transdermalpatches for the delivery of pharmaceutical agents is well known in theart. See, e.g., U.S. Pat. No. 5,023,252. Such patches may be constructedfor continuous, pulsatile, or on demand delivery of pharmaceuticalagents.

In one embodiment, the pharmaceutical composition of the presentinvention is adapted for buccal and/or sublingual, or nasaladministration. This embodiment provides administration of the firstcompound in a manner that avoids gastric complications, such as firstpass metabolism by the gastric system and/or through the liver. Thisadministration route may also reduce adsorption times, providing morerapid onset of therapeutic benefits.

The first compound may be administered over a wide dosage range. Forexample, dosages per day normally fall within the range of about 0.0001to about 30 mg/kg of body weight. In the treatment of adult humans, therange of about 0.1 to about 15 mg/kg/day, in single or divided dose, maybe used. In one embodiment, the formulation comprises about 0.1 mg toabout 1500 mg of a first compound. In another embodiment, theformulation comprises about 1 mg to about 100 mg of a first compound. Inanother embodiment, the formulation comprises about 1 mg to about 50 mgof a first compound. In another embodiment, the formulation comprisesabout 1 mg to about 30 mg of a first compound. In another embodiment,the formulation comprises about 4 mg to about 26 mg of a first compound.In another embodiment, the formulation comprises about 5 mg to about 25mg of a first compound. However, it will be understood that the amountof the first compound actually administered will be determined by aphysician, in the light of the relevant circumstances, including thecondition to be treated, the chosen route of administration, the form ofthe first compound administered, the lipid lowering agent(s)administered, the age, weight, and response of the individual patient,and the severity of the patient's symptoms. Therefore, the above dosageranges are not intended to limit the scope of the invention in any way.In some instances dosage levels below the lower limit of the aforesaidrange may be more than adequate, while in other cases still larger dosesmay be employed without causing any harmful side effect, provided thatsuch larger doses are first divided into several smaller doses foradministration throughout the day.

“Fibrosis” refers to a condition involving the development of excessivefibrous connective tissue, e.g., scar tissue, in a tissue or organ. Suchgeneration of scar tissue may occur in response to infection,inflammation, or injury of the organ due to a disease, trauma, chemicaltoxicity, and so on. Fibrosis may develop in a variety of differenttissues and organs, including the liver, kidney, intestine, lung, heart,etc.

As used herein, a “cholestatic condition” refers to any disease orcondition in which bile excretion from the liver is impaired or blocked,which can occur either in the liver or in the bile ducts. Intrahepaticcholestasis and extrahepatic cholestasis are the two types ofcholestatic conditions. Intrahepatic cholestasis (which occurs insidethe liver) is most commonly seen in primary biliary cirrhosis, primarysclerosing cholangitis, sepsis (generalized infection), acute alcoholichepatitis, drug toxicity, total parenteral nutrition (being fedintravenously), malignancy, cystic fibrosis, biliary atresia, andpregnancy. Extrahepatic cholestasis (which occurs outside the liver) canbe caused by bile duct tumors, strictures, cysts, diverticula, stoneformation in the common bile duct, pancreatitis, pancreatic tumor orpseudocyst, and compression due to a mass or tumor in a nearby organ.

Clinical symptoms and signs of a cholestatic condition include: itching(pruritus), fatigue, jaundiced skin or eyes, inability to digest certainfoods, nausea, vomiting, pale stools, dark urine, and right upperquadrant abdominal pain. A patient with a cholestatic condition can bediagnosed and followed clinically based on a set of standard clinicallaboratory tests, including measurement of levels of alkalinephosphatase, γ-glutamyl transpeptidase (GGT), 5′ nucleotidase,bilirubin, bile acids, and cholesterol in a patient's blood serum.Generally, a patient is diagnosed as having a cholestatic condition ifserum levels of all three of the diagnostic markers alkalinephosphatase, GGT, and 5′ nucleotidase, are considered abnormallyelevated. The normal serum level of these markers may vary to somedegree from laboratory to laboratory and from procedure to procedure,depending on the testing protocol. Thus, a physician will be able todetermine, based on the specific laboratory and test procedure, what anabnormally elevated blood level is for each of the markers. For example,a patient suffering from a cholestatic condition generally has greaterthan about 125 IU/L alkaline phosphatase, greater than about 65 IU/LGGT, and greater than about 17 NIL 5″nucleotidase in the blood. Becauseof the variability in the level of serum markers, a cholestaticcondition may be diagnosed on the basis of abnormal levels of thesethree markers in addition to at least one of the symptoms mentionedabove, such as itching (pruritus).

The term “primary biliary cirrhosis”, often abbreviated PBC, is anautoimmune disease of the liver marked by the slow progressivedestruction of the small bile ducts of the liver, with the intralobularducts (Canals of Hering) affected early in the disease. When these ductsare damaged, bile builds up in the liver (cholestasis) and over timedamages the tissue. This can lead to scarring, fibrosis and cirrhosis.Primary biliary cirrhosis is characterized by interlobular bile ductdestruction. Histopathologic findings of primary biliary cirrhosisinclude: inflammation of the bile ducts, characterized byintraepithelial lymphocytes, and periductal epithelioid granulomata.There are 4 stages of PBC.

Stage 1—Portal Stage: Normal sized triads; portal inflammation, subtlebile duct damage. Granulomas are often detected in this stage.

Stage 2—Periportal Stage: Enlarged triads; periportal fibrosis and/orinflammation. Typically this stage is characterized by the finding of aproliferation of small bile ducts.

Stage 3—Septal Stage: Active and/or passive fibrous septa.

Stage 4—Biliary Cirrhosis: Nodules present; garland

The term “primary sclerosing cholangitis” (PSC) is a disease of the bileducts that causes inflammation and subsequent obstruction of bile ductsboth at a intrahepatic (inside the liver) and extrahepatic (outside theliver) level. The inflammation impedes the flow of bile to the gut,which can ultimately lead to cirrhosis of the liver, liver failure andliver cancer.

The term “Nonalcoholic steatohepatitis” (NASH) is liver inflammationcaused by a buildup of fat in the liver. In some people, the buildup offat causes inflammation of the liver. Because of the inflammation, theliver doesn't work as well as it should. NASH can get worse and causescarring of the liver, which leads to cirrhosis. NASH is similar to thekind of liver disease that is caused by long-term, heavy drinking. ButNASH occurs in people who do not abuse alcohol.

The term “organ” refers to a differentiated structure (as in a heart,lung, kidney, liver, etc.) consisting of cells and tissues andperforming some specific function in an organism. This term alsoencompasses bodily parts performing a function or cooperating in anactivity (e.g., an eye and related structures that make up the visualorgans). The term “organ” further encompasses any partial structure ofdifferentiated cells and tissues that is potentially capable ofdeveloping into a complete structure (e.g., a lobe or a section of aliver).

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the description and examples providedherein are for purposes of illustration and not limitation of the claimsthat follow.

In the specification, the singular forms also include the plural, unlessthe context clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In the case of conflict, the present specificationwill control. All percentages and ratios used herein, unless otherwiseindicated, are by weight.

EXAMPLES Example 1: Bile Duct Ligation (BDL) Model

This experiment was performed to evaluate the effects of OCA andatorvastatin alone, and in combination, on fibrosis induced by commonbile duct ligation in mice.

Animals, Housing and Diet

Male C57BL/6 mice (6 weeks of age) were obtained from Japan SLC. Theanimals were maintained in a specific pathogen free facility undercontrolled conditions of temperature (23±2° C.), humidity (45±10%),lighting (12-hour artificial light and dark cycles; light from 8:00 to20:00), and air exchange (air exchange rate: more than 40 times/hour). Ahigh pressure (20±4 Pa) was maintained in the experimental room toprevent contamination of the facility. The animals were housed in KN-600(Natsume Seisakusho, Japan) with a maximum of 6 mice per cage.Sterilized Paper-Clean (Japan SLC) was used for bedding and replacedonce a week. Sterilized solid high fat diet (HFD) and water wereprovided ad libitum for 3 weeks before the day of surgery.

Treatment Groups Group 1: Sham

-   -   Sham-operated mice (n=8) were orally administered vehicle        (0.5% CMC) in a volume of 5 mL/kg once daily from day 0 to day 6        after BDL surgery.

Group 2: BDL-Vehicle

-   -   BDL-operated mice (n=12) were orally administered vehicle        (0.5% CMC) in a volume of 5 mL/kg once daily from day 0 to day 6        after BDL surgery.

Group 3: BDL-OCA

-   -   BDL-operated mice (n=12) were orally administered vehicle        supplemented with OCA at a dose of 5 mg/kg once daily from day 0        to day 6 after BDL surgery.

Group 4: BDL-Atorvastatin

-   -   BDL-operated mice (n=12) were orally administered vehicle        supplemented with atorvastatin at a dose of 10 mg/kg once daily        from day 0 to day 6 after BDL surgery.

Group 5: OCA-BDL-Atorvastatin

-   -   BDL-operated mice (n=12) were orally administered vehicle        supplemented with OCA at a dose of 5 mg/kg and atorvastatin at a        dose of 10 mg/kg once daily from day 0 to day 6 after BDL        surgery.

Bile Duct Ligation Surgery

Bile duct ligation surgery was performed at day 0. Cholestasis, whichleads to fibrosis of the liver over time, was established in the mice bythe ligation of the common bile duct under pentobarbital anesthesia.Mice were divided into two surgical cohorts based on their weight beforethe day of surgery. After shaving the hair, the abdominal cavity wasopened and the common bile duct was ligated twice with 5-0 surgical silkand the common bile duct was cut between the ligatures. The peritoneumand the skin were closed with sutures. The mice were transferred to aclean cage (resting cage) for recovery from anesthesia. Sham mice wereoperated in a similar manner to other groups but the bile duct notligated.

Animal Monitoring and Sacrifice

Viability, clinical signs and behavior were monitored daily. Body weightwas recorded daily during the treatment period. Food consumption wasmeasured twice weekly per cage during the treatment period. At day 6,the animals were sacrificed by exsanguination through the direct cardiacpuncture under ether anesthesia (Wako Pure Chemical Industries).

Histological Analysis

To visualize collagen deposition, Bouin's-fixed left lateral liversections were stained using picro-Sirius red solution (Waldeck,Germany). For quantitative analysis of the fibrosis area, bright fieldimages of Sirius red-stained sections were captured around the centralvein using a digital camera (DFC295; Leica, Germany) at 100-foldmagnification, and the positive areas in 5 fields/section were measuredusing ImageJ software (National Institute of Health, USA). Statisticalanalyses were performed using Prism Software 6 (GraphPad Software, Inc.USA).

Results

Histopathological analyses were performed on liver sections (accordingto routine methods) by Sirius-red staining to estimate the percentage offibrosis area. Representative photomicrographs of Sirius red-stainedliver sections are shown in FIGS. 1A-1E. The BDL-Vehicle group showed asignificant increase in Sirius red-positive area compared with theBDL-Sham group. As indicated in Table 1 and FIG. 2, theBDL-OCA+atorvastatin group showed a significant decrease in Siriusred-positive area compared with the BDL-Vehicle group.

TABLE 1 BDL- Parameter BDL- BDL- BDL- OCA + (mean ± Sham Vehicle OCA ATOATO SD) (n = 8) (n = 11) (n = 12) (n = 12) (n = 12) Sirius red- 1.30 ±0.45 2.17 ± 0.43 2.06 ± 0.37 2.31 ± 0.63 1.60 ± 0.44 positive (p < 0.05)area (%)

The results in Table 1 indicate that the combination of OCA andatorvastatin significantly reduced fibrosis.

Example 2: Diet Induced Nash in Apoe*3Leiden.Cetp Mice

This experiment was performed to evaluate the effects of OCA andfenofibrate, alone or in combination, on the development of diet inducedNASH and liver fibrosis in APOE*3Leiden.CETP transgenic mice. Hepaticgene expression profiling and subsequent pathway analysis were performedto determine whether the combination regulates novel genes not regulatedby either monotherapy treatment, and/or more strongly regulates genesalso impacted by the monotherapy.

Animals, Housing and Diet

Male APOE*3Leiden.CETP transgenic mice (9-21 weeks old) were obtainedand housed 2-5 mice per cage. The mice were fed a high fat dietcontaining 24% lard and 1% (w/w) cholesterol. The run in period was 15weeks on the high fat diet. At week 16, mice were matched based on age,body weight, plasma cholesterol and triglycerides after 4 h fasting.

Treatment Groups Group 1: HFC Reference Group Start Treatment

-   -   Mice (n=15) were fed a high fat diet during the run in weeks 0        to 14.        Group 2: HFC control group    -   Mice (n=15) were fed a high fat diet from weeks 0 to 24.

Group 3: HFC+OCA

-   -   Mice (n=15) were fed a high fat diet supplemented with OCA at a        dose of 10 mg/kg once daily from week 16 to 24.        Group 4: HFC+low dose fenofibrate    -   Mice (n=15) were fed a high fat diet supplemented with        fenofibrate at a dose of 10 mg/kg once daily from week 16 to 24.        Group 5: HFC+high dose fenofibrate    -   Mice (n=15) were fed a high fat diet supplemented with        fenofibrate at a dose of 40 mg/kg once daily from week 16 to 24.        Group 6: HFC+OCA+low dose fenofibrate    -   Mice (n=15) were fed a high fat diet supplemented with OCA at a        dose of 10 mg/kg once daily and fenofibrate at a dose of 10        mg/kg once daily from week 16 to 24.        Group 7: HFC+OCA+high dose fenofibrate    -   Mice (n=15) were fed a high fat diet supplemented with OCA at a        dose of 10 mg/kg once daily and fenofibrate at a dose of 40        mg/kg once daily from week 16 to 24.        Group 8: Chow control group    -   Mice (n=8) were feed chow from week 0 to 24.

Study Design

Mice were fed a high fat chow (HFC) diet for 14 weeks. After 15 weeks onthe HFC diet, HFC mice were matched into 7 groups based on age, bodyweight, plasma cholesterol and triglycerides after 4 h fasting. Micewere treated with of OCA and fenofibrate, alone or in combinationbeginning at week 15 and sacrificed at week 25 in an unfasted state. Oneweek before sacrifice, mice were labeled with D₂O, by i.p. injection ofa bolus of D₂O and subsequent addition of 4% D₂O to the drinking water.Plasma (EDTA) was obtained by heart puncture and stored at −70° C. Theliver was weighed and 4 pieces of liver were isolated: 1 piece (mediallobe) was fixed in 10% formalin (for NASH and fibrosis histology) and 3pieces (sinister lobe) were snap-frozen in liquid N₂ and storedindividually at −70° C.

Hepatic Inflammation Score

Inflammation is a key feature of NASH. Inflammation was categorizedaccording to the procedure by Liang et al., PlosOne 2014 Dec., 9(12) andscored by quantitatively analyzing the number of inflammatory cellsaggregates. In particular, the level of inflammation was evaluated bycounting the number of inflammatory foci per field using a 100×magnification (view size of 3.1 mm2; average of five different fields).

Results: Summary of Effects of OCA +/− Fenofibrate on Inflammation inAPOE*3-Leiden.CETP Mice

The effects of OCA 10 mg/kg were investigated alone and in combinationwith fenofibrate (10 and 40 mg/kg) on inflammation in APOE*3-Leiden.CETPmice on a NASH diet. After 10 weeks of drug administration at the lowdose, neither OCA (10 mg/kg) nor fenofibrate (10 mg/kg) reduced thenumber of inflammatory cell foci (FIGS. 3A and 3B and Table 2). Bycontrast, the combination significantly decreased inflammation relativeto the vehicle control as well as each monotherapy arm. A higher dose offenofibrate (40 mg/kg/d) also significantly reduced inflammationrelative to vehicle controls. When combined with OCA, no additionalanti-inflammatory effects were evident as the high dose of fenofibrateexerted a near-maximal effect on its own. In summary, a significantreduction in inflammation with the OCA+low dose fenofibrate combination(−63%) was observed. Furthermore, a significant reduction with the highdose of fenofibrate (−74%) and a similar reduction in combination withOCA (−79%) were observed. See Table 2 and FIGS. 3A and 3B.

TABLE 2 Inflammation Group (# of inflammatory cell foci) Group 1: HFCreference group 24.3 ± 17.3 Group 2: HFC control group 27.5 ± 18.0  (n =15) Group 3: OCA 29.1 ± 22.7 Group 4: low dose fenofibrate 22.0 ± 15.6Group 5: high dose fenofibrate 7.1 ± 5.9 Group 6: OCA + low dosefenofibrate 10.0 ± 7.0  Group 7: OCA + high dose fenofibrate 5.1 ± 7.0Group 8: Chow control 0.8 ± 0.4

The results in Table 2 suggests that the efficacy of the combination ofOCA and the high dose of fenofibrate is driven and reaches an upperlimit by the high dose of fenofibrate.

RNA Isolation and Sequencing

Nucleic acid extraction was performed as described previously in detail(Verschuren et al., 2014). Briefly, total RNA was extracted fromindividual liver samples using glass beads and RNAzol (CamproScientific, Veenendaal, The Netherlands). RNA concentration and qualitywas determined using the Fragment Analyzer (Advanced AnalyticalTechnologies, USA) and the RNA 6000 Nano Lab-on-a-Chip kit and aBioanalyzer 2100 (Agilent Technologies, Amstelveen, The Netherlands).All samples met the quality requirements and were used in the RNAsequencing procedure.

The NEBNext Ultra Directional RNA library Prep Kit for Illumina was usedto process the samples. The sample preparation was performed accordingto the protocol “NEBNext Ultra Directional RNA Library Prep Kit forIllumina” (NEB #E7420S/L). Briefly, mRNA was isolated from total RNAusing oligo-dT magnetic beads. After fragmentation of the mRNA, a cDNAsynthesis was performed. This was used for ligation of sequencingadapters and PCR amplification of the resulting product. The quality andyield after sample preparation was measured with the Fragment Analyzer(Advanced Analytical Technologies, USA). The size of the resultingproducts was consistent with the expected size distribution (a broadpeak between 300-500 bp).

Clustering and DNA sequencing using the Illumina NextSeq 2500 wasperformed according manufacturer's protocols. Data was generated usingsingle-end read sequencing protocol obtaining approx. 15 million readsper sample and 75 bp per read. Image analysis, base calling, and qualitycheck was performed with the Illumina data analysis pipeline RTA v2.4.11to generate the raw data (*.fastq-files).

The reads were mapped to the reference sequence Mus musculus GRCm38.p3using a short read aligner based on Burrows-Wheeler Transform. Thedefault mismatch rate of 2% (3 mismatches in a read of 150bases) wasused. Based on the mapped read locations in the alignment files(*.bam-files) the frequency of how often a read was mapped on atranscript was determined. The counts were saved to count-files, whichserved as input for downstream mRNA-seq differential expressionanalysis. The read counts were loaded into the DESeq package, astatistical package within the R platform. DESeq was specificallydeveloped to normalize RNA-seq data for different samples and finddifferentially expressed genes between two conditions for RNA-seq datato estimate the relationship between the mean and variance of each gene(Anders et al., 2013). Furthermore, it allows scaling factors to beeasily included in the statistical test. Differentially expressed geneswere identified using a threshold for significance of P<0.01 and geneswere used as an input for pathway analysis through Ingenuity PathwayAnalysis (IPA) suite (accessed 2016).

Upstream regulator analysis was performed using the IPA software (Krameret al., 2014). This analysis determines the activation state oftranscription factors based on the observed differential geneexpression. This results in an overlap P-value and activation z-scorefor each transcription factor in the IPA knowledge base. The overlapP-value indicates the significance of the overlap between the knowntarget genes of a transcription factor and the differentially expressedgenes measured in an experiment. The activation z-score indicatesactivation (positive z-score) or inhibition (negative z-score) of aparticular transcription factor. An activation z-score <−2 or >2indicates significant inhibition or activation of a pathway or process.

Omics Results

Next Generation Sequencing was performed on liver mRNA samples fromtreated mice to gain insight into the underlying mechanisms andpathways. Two analyses were performed to gain insight into theunderlying mechanisms and pathways.

First, an enrichment analysis of the canonical pathways analysisrevealed that OCA regulated several inflammatory processes (FIG. 6A).The figure plots each pathway as a function of −log p-value (forreference a transformed value for p<0.05 is 1.3, p<0.0001 is 4, forp<0.000005 is 5.3 etc.). The regulated pathways with OCA monotherapywere related to T- and B-cell signing, leukocyte extravasationsignaling, natural killer cell signaling etc. The low dose offenofibrate had no effect on these pathways. When OCA was combined withthe low dose of fenofibrate, some of the same pathways were stronglyregulated (in the case of iCOS-iCOSL signaling in T-helper cells,leukocyte extravasation signaling, pattern recognition receptors, FCreceptor-mediated phagocytosis in macrophages, and phagosome formation).Additionally, other pathways (e.g., cholesterol biosynthesis I and II,fatty acid b-oxidation) that were not significantly regulated by eitheragent alone were regulated by the combination (FIG. 6B). As with thehistological data, the high dose of fenofibrate had a robust effect onthese pathways but was not enhanced with OCA co-administration.

A more detailed analysis was performed around the pathways involved inleukocyte extravasation signaling. Extravasation of leukocytes isessential for pathophysiological processes in NASH (and other diseases).These processes include migration of T-lymphocytes for immunesurveillance, recruitment of activated lymphocytes and granulocytesduring acute and chronic inflammatory responses, and homing andmobilization of hematopoietic progenitor cells. The effects ofmaintaining mice on a high-fat diet from the study illustrates theseprocesses in which significantly upregulated and down-regulated genes.Table 3 describes the effects of a high-fat diet on leukocyteextravasation signaling in mice maintained on a high-fat diet relativeto mice maintained on standard chow, and also the effects of combinationtreatment relative to high fat diet.

TABLE 3 Significantly Significantly regulated by regulated by NASH DietCombo vs. Gene Full Name/s Predicted Function vs. CHOW NASH DIET CD43Cluster of differentiation Major sialoglycoprotein located ↑ ↓ 43 orleukosialin on the surface of T lymphocytes, monocytes, granulocytes andsome B lymphocytes. CD44 Cluster of differentiation Cell-surfaceglycoprotein ↑ ↓ 44 involved in cell-cell interactions, cell adhesionand migration CDH5 Cadherin 5 or CD144 Imparts cells the ability toadhere ↑

in a hemophilic manner and controls cohesion and organization ofintercellular junctions CRK CT10 regulator of kinase Adapter protein inintracellular ↓

or p38 signaling pathways CXCR4 Chemokine C-X-C Receptor for chemotacticactivity ↑ ↓ Motif Receptor 4 or for lymphocytes CD184 ERM Exrin,Radixzin, Moesin Crosslinks actin filaments with ↓ ↓ protein familyplasma membranes EPAC Exchange protein Intracellular sensors for cAMP ↓

activated by cAMP ICAM-1 Intracellular adhesion Cell surfaceglycoprotein that ↑ ↓ molecule 1 or CD54 binds integrins ITGA4 Integrinalpha subunit Large subunit of a4b1 ↑ ↓ lymphocyte homing receptor ITGALIntegrin alpha L or Cellular adhesion and ↑ ↓ CD11A costimulatorysignaling ITGAM Integrin Alpha M or Regulates leukocyte adhesion ↑ ↓CD11B and migration ITGB1 Integrin Beta-1 or CD29 Integrins participatein cell ↓

adhesion and cell-surface mediated signaling ITGB2 Integrin Beta-2 orCD18 Integrins participate in cell ↑ ↓ adhesion and cell-surfacemediated signaling JAM2 Junction adhesion Adhesive ligand forinteracting ↑

molecule 2 or CD322 with multiple immune cell types and lymphocytehoming JAM3 Junction adhesion Binds with JAM2 in the ↓

molecule 3 regulation of adhesion LFA-1 Lymphocyte function- Adhesionmolecule on T-cells,

↓ associated antigen 1 B-cells, macrophages and neutrophils NCF1Neutrophil cytosolic A subunit of the neutrophil ↑ ↓ factor-1 NADPHoxidase NCF2 Neutrophil cytosolic A subunit of the neutrophil ↑ ↓factor-2 NADPH oxidase NCF4 Neutrophil cytosolic A subunit of theneutrophil ↑ ↓ factor-4 NADPH oxidase NOX NADPH oxidase Enzymes thattransport electrons ↑ ↓ across plasma membrane and generate superoxidesand downstream reactive oxygen species PECAM1 Platelet endothelial cellLeukocyte transmigration, ↑

adhesion molecule or angiogenesis, and integrin CD31 activation PKCProtein kinase C Part of family of enzymes

controlling phosphorylation of serine and threonine amino acids on otherproteins PI3K PI3-kinases PI3Ks are a family of related

↓ intracellular signal transducer enzymes PLC Phospholipase C catalyzesthe formation of

↓ inositol 1,4,5 -trisphosphate and diacylglycerol fromphosphatidylinositol 4,5 - bisphosphate PSGL-1 P-selectin GlycoproteinRole in leukocyte trafficking ↑ ↓ Ligand 1 during inflammation bytethering of leukocytes to activated platelets or endothelia expressingselectins Rac2 Ras-related C3 Regulates diverse cellular events ↑ ↓botulinum toxin including growth, cytoskeletal substrate reorganizationand activation of protein kinases RASGRP 1 RAS guanyl nucleotide-Activates Erk/MAP kinase ↑ ↓ releasing protein cascade and regulates T-and B- cell development, homeostasis and differentiation RhoH Rashomolog gene H Regulates intracellular actin ↑ ↓ dynamics RhoGAP RHOGTPases Protein domain of GTPase

↓ activating proteins SPA-1 Signal-induced May hamper mitogen-induced ↑

proliferation associated cell cycle progression when protein 1abnormally expressed THY-1 Thymocyte Cell-cell and cell-matrix ↑ ↓differentiation antigen 1 interactions, may impact neurite or CD90outgrowth, nerve regeneration, apoptosis, metastasis, inflammation, andfibrosis TIMP Tissue inhibitor of Bind and inactivate tissue

↓ metalloproteinase metalloproteinases VASP Vasodilator-stimulatedInvolved in intracellular ↑ ↓ phosphoprotein signaling pathways thatregulate integrin-extracellular matrix interactions VAV VAV Aprotooncogene mediating antigen-induced activation of B ↑ ↓ lymphocytesVCAM1 Vascular cell adhesion Adhesion of lymphocytes, ↑ ↓ protein 1 orCD 106 monocytes, eosinophils, and basophils to vascular endotheliumWASP Wiskott-Adlrich Important in leukocyte motility

↓ Syndrome) in vivo

Transmigration and extravasation of leukocytes across the endotheliumoccurs in several distinct steps including rolling of the leukocytesover the endothelial cells, mediated by transient weak interactionsbetween adhesion molecules. Subsequently, loosely attached leukocytesare in such close proximity of the endothelium that they are activatedby chemotactic cytokines, presented on the apical surface of theendothelium. Next, activated leukocytes spread and firmly adhere to theendothelium forming docking structures and ultimately migrate throughthe intercellular clefts between the endothelial cells to the underlyingtissue.

The administration of OCA downregulates numerous genes involved in thisprocess of the inflammatory cascade within the leukocyte (WAP, Rac2,RASGRP1, Vav, PKC, PI3K, ERM, ITGAL and PSGL-1) as well as withinendothelial cells (VCAM1, PI3K, ERM, NOX, CYBA, PKC, NCF1 and 2). Generegulation within these pathways was not evident followingadministration of a low dose of fenofibrate alone.

When OCA was combined with a dose of fenofibrate that was ineffective atregulating these pathways, multiple additional genes are now regulatedpointing to a synergistic effect. Within the leukocyte these additionalgenes included CD43, PSGL-1, CXCR4, ITGAM, ITGB2, Rap1, ITGA4. Withinthe endothelial cell these additional genes included ICAM1, RhoGAP,VASP, NCF4, ITGAM, ITGB2, ITGA4 and ICAM-1.

As noted above, the high dose of fenofibrate had numerous effects onthis pathway that were not enhanced with OCA co-administration. Allsubsequent analyses focused on the low dose monotherapies andcombination (OCA 10 mg/kg+/−fenofibrate 10 mg/kg).

In a second analysis, the genes differentially regulated between the lowdose combination and each respective monotherapy were compared. Thisdiffers from the first gene expression analyses (described above) whichfocused on comparisons relative to the vehicle group; the analysis belowcompares each monotherapy to the combination. The Venn diagram (FIG. 7A)shows that OCA has 109 uniquely regulated genes, fenofibrate has 92uniquely regulated genes and 6 commonly regulated genes. The combinationregulated 517 overlapping genes with OCA, 75 with fenofibrate, and 5genes were common to all. Of note, the combination regulated a total of912 unique genes. A subsequent pathway enrichment highlights thebiological processes in which the combination genes are involved (FIG.7a ).

Subsequent pathway analyses were conducted both for leukocyteextravasation (e.g., as above but this time comparisons are between thecombination and each monotherapy. For leukocyte extravasation,comparisons of the combination versus each monotherapy revealed thatthere were a number of uniquely regulated genes consistent with theobserved enhanced anti-inflammatory changes noted histologically in thecombination-treated mice (Table 4).

TABLE 4 Combination Combination vs. Fenofibrate vs. OCA Gene Full Name/sPredicted Function Monotherapy Monotherapy CD44 Cluster ofdifferentiation 44 Cell-surface glycoprotein ↓ involved in cell-cellinteractions, cell adhesion and migration CXCR4 Chemokine C-X-C MotifReceptor for chemotactic activity ↓ Receptor 4 or CD184 for lymphocytesCYBA1 Cytochrome b(-245) Encodes alight chain of ↓ component cytochromeb(-245) which is a of the NOX complex ERM Exrin, Radixzin, MoesinCrosslinks actin filaments with ↓ protein family plasma membranes ICAM-1Intracellular adhesion Cell surface glycoprotein that ↓ ↓ molecule 1 orCD54 binds integrins ITGA4 Integrin alpha subunit Large subunit of a4b1↓ ↓ lymphocyte homing receptor ITGAL Integrin alpha L or CD11A Cellularadhesion and ↓ costimulatory signaling ITGAM Integrin Alpha M orRegulates leukocyte adhesion ↓ CD11B and migration ITGB2 Integrin Beta-2or CD18 Integrins participate in cell ↓ ↓ adhesion and cell-surfacemediated signaling LFA-1 Lymphocyte function- Adhesion molecule onT-cells, ↓ ↓ associated antigen 1 B-cells, macrophages and neutrophilsMMP9 Matrix metalloprotease 9 Degrades collagen of the ↓ extracellularmatrix NCF1 Neutrophil cytosolic factor- A subunit of the neutrophil ↓ 1NADPH oxidase NCF2 Neutrophil cytosolic factor- A subunit of theneutrophil ↓ 2 NADPH oxidase NCF4 Neutrophil cytosolic factor- A subunitof the neutrophil ↓ 4 NADPH oxidase NOX NADPH oxidase Enzymes thattransport electrons ↓ across plasma membrane and generate superoxidesand downstream reactive oxygen species PKC Protein kinase C Part offamily of enzymes ↓ controlling phosphorylation of serine and threonineamino acids on other proteins PI3K PI3-kinases PI3Ks are a family ofrelated ↓ ↓ intracellular signal transducer enzymes PLC□ Phospholipase Ccatalyzes the formation of ↓ inositol 1,4,5-trisphosphate anddiacylglycerol from phosphatidylinositol 4,5-bisphosphate PSGL-1P-selectin Glycoprotein Role in leukocyte trafficking ↓ ↓ Ligand 1during inflammation by tethering of leukocytes to activated platelets orendothelia expressing selectins Rac2 Ras-related C3 botulinum Regulatesdiverse cellular events ↓ toxin substrate including growth, cytoskeletalreorganization and activation of protein kinases Rap 1GAP RAP 1GTPase-activating RAP1 is of particular interest ↓ protein 1 since ithas been shown to be an antagonist of RAS and is capable of suppressingcellular transformation RASGR RAS guanyl nucleotide- Activates Erk/MAPkinease ↓ P1 releasing protein cascade and regulates T- and B-celldevelopment, homeostasis and differentiation RhoH Ras homolog gene HRegulates intracellular actin ↓ dynamics RhoGAP RHO GTPases Proteindomain of GTPase ↓ activating proteins TIMP Tissue inhibitor of Bind andinactivate tissue ↓ metalloproteinase metalloproteinases VASPVasodilator-stimulated Involved in intracellular ↓ phosphoproteinsignaling pathways that regulate integrin-extracellular matrixinteractions VAV VAV A protooncogene mediating ↓ antigen-inducedactivation of B lymphocytes VCAM1 Vascular cell adhesion Adhesion oflymphocytes, ↓ protein 1 or CD 106 monocytes, eosinophils, and basophilsto vascular endothelium WASP Wiskott-Adlrich Syndrome) Important inleukocyte motility ↓ in vivo

Given the progression from inflammation to fibrosis in NASH, and theobservation that hepatic fibrosis/HSC pathways emerged as significantlyregulated in the combination we also examined pathways in HSCs. Whencompared against the monotherapy, it is clear that more genes areregulated in the combination versus fenofibrate alone and fewer genesare regulated in the combination versus OCA. In other words, withrespect to these fibrotic pathways, there is clearly an interactionbetween both agents, but the OCA portion of the combination may be morestrongly driving these effects.

Interpretation and Relevance

The importance of FXR activation in preventing fibrosis and inflammationis demonstrated in livers from FXR knockout mice which display elevatedexpression of inflammatory genes (Kim 2007) with progressive age-relatedinjury and inflammation (Yang 2007). Consistent with these reports, OCAexerted anti-inflammatory properties in HepG2 cells and mouse primaryhepatocytes. HepG2 cells pretreated with OCA and then exposed topro-inflammatory stimuli exhibited a 50% to 60% reduction in TNF-α mRNAlevels, cyclo-oxygenase-2 (COX-2) induction and TNF-α-stimulatedinducible nitric oxide synthase (iNOS) expression. Likewise, OCA-treatedprimary hepatocytes displayed a blunted induction (by 40% to 50%) ofiNOS and monocyte chemoattractant protein-1 (MCP-1) gene expression inresponse to pro-inflammatory stimuli (Wang 2008). The effects of OCA onmechanisms of cell migration have not been studied directly, however OCAinhibited the production of iNOS or COX-2 induced by IL-1β and abolishedpharmacologically-induced rat aortic smooth muscle cell migration (Li2007). Similar inhibition of inflammatory infiltrates with OCA has beendemonstrated in the intestinal tissue of two animal models ofinflammatory bowel disease (DSS and trinitrobenzene sulfonic acid)(Gadaleta 2011) and the kidney of a rat model of Type 1 diabetes (Wang2010). Thus, the observation of enhanced anti-inflammatory effects byOCA suggests that changes in gene expression with OCA in combinationwith fenofibrate could enhance the inhibition of inflammation andinflammatory cell migration in a number of disease conditions.

Example 3: Diet Induced NASH in Leptin-Deficient Ob/Ob Mice

This experiment was performed to evaluate the effect of 8 weeks oftreatment with OCA and atorvastatin alone and in combination on thefibrosis stage (pre-biopsy vs. post-biopsy) in male leptin-deficientob/ob-NASH mice

Animals, Housing and Diet

Male Lep^(ob)/Lep^(ob) mice (at 5 weeks of age) were purchased fromJanVier, France. During the acclimatization and diet-induction period,the mice were group housed five per cage in custom-made cabinets under a12:12 light dark cycle (lights on from 3AM-3PM) at controlledtemperature conditions (22±1° C.; 50±10% relative humidity). Throughoutthe diet induction and study period, the mice had ad libitum access tocustom made NASH diet (S8189, Ssniff, Germany) (40% fat, 40%carbohydrates (20% fructose) and 2% cholesterol) or regular rodent chow(ob/ob-CHOW) (Altromin 1324, Brogaarden, Denmark), and tap water. Theanimals were kept on the diet for a total of 18 weeks beforeintervention and maintained on the diet throughout the study period.Animals were singly-housed during post-operative recovery and throughoutthe study period.

Treatment Groups

Group 1: Lep^(ob)/Lep^(ob)-NASH Vehicle

-   -   Mice (n=10) were orally administered vehicle (0.5% CMC) in a        volume of 5 mL/kg once daily from week 0 to 8.        Group 2: Lep^(ob)/Lep^(ob)-NASH OCA    -   Mice (n=10) were orally administered vehicle supplemented with        OCA at a dose of 30 mg/kg once daily from week 0 to 8.        Group 3: Lep^(ob)/Lep^(ob)-NASH    -   Mice (n=11) were orally administered vehicle supplemented with        Atorvastatin at a dose of 10 mg/kg once daily from week 0 to 8.        Group 4: Lep^(ob)/Lep^(ob)-NASH OCA+Atorvastatin    -   Mice (n=9) were orally administered vehicle supplemented a        combination of with OCA at a dose of 30 mg/kg and Atorvastatin        at a dose of 30 mg/kg once daily.        Allocation into Studies, Stratified Randomization and Baseline        Monitoring

After 15 weeks of diet induction (3 weeks prior to study start), a liverbiopsy was obtained assessment of hepatic progression of fibrosis andsteatosis, and for liver Fibrosis Stage evaluation. At week −1, astratified randomization into treatment groups was performed accordingto liver fibrosis stage, steatosis score, and body weight.

Pre-Biopsy Procedure

On the day of the operation, mice were anesthetized with isoflurane(2-3%) in 100% oxygen. A small abdominal incision in the midline wasmade and the left lateral lobe of the liver was exposed. A cone shapedwedge of liver tissue (˜100 mg) was excised from the distal portion ofthe lobe, weighed, and fixated in 4% paraformaldehyde (PFA) forhistology. The cut surface of the liver was instantly electrocoagulatedusing bipolar coagulation (ERBE VIO 100 electrosurgical unit). The liverwas returned to the abdominal cavity and the abdominal was sutured andthe skin was closed with staplers. On the day of operation, micereceived warmed saline (0.5 ml) for rehydration. For post-operationrecovery, carprofen (5 mg/ml-0.01 ml/10 g) and enrofloxazin (5 mg/ml-1ml/kg) were administered subcutaneously on the day of operation andpost-operation days 1 and 2.

Pre-Screening for Assessment of Hepatic Level of Steatosis and Fibrosis

Liver biopsy preparation for histological assessment: After overnightstorage in 4% PFA, liver biopsies were infiltrated overnight in paraffinin an automated Miles Scientific Tissue-TEK VIP Tissue Processor andsubsequently embedded in paraffin blocks. Biopsies from five differentanimals were embedded on one block. The blocks were then trimmed and two3 μm sections were cut (one for Sirius Red and one for H&E staining) ona Microm HM340E Microtome (Thermo Scientific). Two blocks were placed onone slide giving a total of 10 biopsies per slide representing 10different animals. Sections were left to dry over-night. Evaluation offibrosis stage for stratification and randomization into treatmentgroups were performed as outlined by Kleiner et al. (2005) (see below).

Baseline and Final Plasma Biomarkers

Blood samples for measuring non-fasting (fed) plasma levels oftriglycerides were obtained in the morning (7-8AM) at baseline and inweek 8 of treatment. The blood samples were collected from the tail vein(by snipping) in a conscious state. The latest drug dose wasadministered ˜18 hours before blood sampling. Mice were re-fed after theblood sampling.

Termination and Necropsy

Animals were terminated in week 8 in a non-fasting state. Latest drugdose was administered ˜18 hours before termination and animals will notreceive drug dosing prior to termination. Animals were induced by CO₂/O₂and during anesthesia (isoflurane), the abdominal cavity is opened andcardiac blood obtained for collection of terminal plasma. Upon necropsy,whole liver was collected and weighed. A biopsy from the left laterallobe was excised and fixated in 4% PFA for histology and biochemicalanalysis. The median lobe was divided into pieces and snap frozen inliquid nitrogen for biochemical analysis (TG). Remaining liver tissuewas subsequently fixated in 4% PFA for later optional histology.

Liver Tissue Processing

Pre-study biopsy: Approximately three weeks before study start, a coneshaped wedge of liver tissue (˜100 mg) was excised from the distalportion of the left lateral lobe, weighed and immediately placed in 4%PFA.

Terminal liver tissue: Following 8 weeks of treatment, the whole liverwas collected, weighed and liver biopsy from the left lateral lobe isexcised and immediately placed in 4% PFA (˜150-200 mg). Pieces of medianlobe will be snap frozen in cryotubes (RNAseq)(˜100 mg) and in FastPreptubes for TG (˜100 mg) and for TC (˜50 mg).

Fixation, embedment and sections for histology: Following an over-nightfixation in 4% PFA, liver biopsies were infiltrated over-night inparaffin in an automated Miles Scientific Tissue-TEK VIP TissueProcessor and subsequently embedded in paraffin blocks. Biopsies fromfive different animals were embedded in one block. The blocks weretrimmed and two 3 μm sections per block were cut on a Microm HM340EMicrotome (Thermo Scientific). One section from two different blocks wasplaced on one object slide giving a total of 10 biopsies per slide asoutlined above.

Fibrosis Stage

Liver pre-biopsy and post-biopsy tissue from the left lateral lobe wascollected for assessment of fibrosis stage by use of clinical criteriaoutlined by Kleiner and colleagues (Design and validation of ahistological scoring system for nonalcoholic fatty liver disease,Kleiner et al, Hepatology 41; 2005) and reproduced in Table 5 below.FIG. 4 describes the effect OCA and atorvastatin alone and incombination on fibrosis scoring. The combination of OCA and atorvastatinshows a trend in lowering the fibrosis score although not in asignificantly manner from vehicle (p value=0.09).

TABLE 5 Feature Degree Score Fibrosis None 0   Perisinusoidal orperiportal 1   Mild, zone 3, perisinusoidal 1A Moderate, zone 3, 1Bperisinusoidal Portal/periportal 1C Perisinusoidal & 2  portal/periportal Bridging fibrosis 3   None 0  

Plasma Triglycerides

Triglyceride Levels: 100 μl blood is collected into Lithium-Heparintubes. Plasma was separated and samples were be stored at −80 degreesCelsius until analysis. Triglyceride levels were measured in singledeterminations using autoanalyzer Cobas C-111 with commercial kit (RocheDiagnostics, Germany) according to the manufacturer's instructions. Asindicated in FIGS. 5A and 5B, the combination of OCA and atorvastatinreduced triglyceride levels in a significantly statistically manner.

Example 4: Sandwich Culture of Hepatocytes

This experiment will be performed to evaluate the effect of OCA incombination with a PPAR agonist or statin to determine their ability toalter collagen synthesis in human hepatocytes.

Reagents and Solutions

Suitable cell culture medium includes Waymouth's MB-752/1, Ham's F12,RPMI 1640, Dulbecco's modified Eagle's medium, Williams' medium E,Leibovitz′ L15 and modified Chee's medium. Type IV collagenase, type Icollagen, Percoll, culture medium and supplements are added to theculture medium (e.g., serum, antibiotics, amino acids, hormones such asDEX, insulin, and growth factors), perfusion buffer, and other solutionswere commercially available or made from commercially availablematerials. Other types of collagen (types II-IV), laminin, fibronectin,and heparin sulfate proteoglycans can be used in the sandwich hepatocyteculture. However, it has been shown that type I and IV collagen weresuperior to fibronectin and laminin.

Isolation of Hepatocytes

A two-step in situ collagenase perfusion method will be utilized toisolate hepatocytes. Briefly, hepatocytes will be isolated from femaleLewis rats. Animals will be anesthetized. The liver will be firstperfused through the portal vein in situ with a perfusion buffer. Theperfusate will be equilibrated before entering the liver. The liver willbe subsequently perfused with collagenase in the perfusion buffer. Theliver will then dissected and transferred to ice-cold perfusion buffer.The liver capsule will be teased apart, and the resulting cellsuspension will be filtered. The cell pellet will be collected bycentrifugation and resuspended. Percoll will be added to the suspension,and hepatocytes separated using a Percoll density centrifugationtechnique. The mixture will be centrifuged, and the cell pellet washedtwice with medium. Hepatocytes viability will be determined by Trypanblue exclusion. Alternatively, cryopreserved hepatocytes can be usedinstead of freshly isolated hepatocytes.

Sandwich Culture of Hepatocytes

Isolated hepatocytes will be cultured on collagen-coated tissue cultureplates and maintained in culture medium supplemented with serum,penicillin, streptomycin, epidermal growth factor, insulin, glucagon andhydrocortisone. A collagen gelling solution will be prepared by mixingType I collagen solution and culture medium. Tissue culture plates willbe coated with the gelling solution and incubated at 37° C. to promotegel formation. Hepatocytes will be seeded at a proper density andmaintained at 37° C. The culture medium will be replaced every 24 hours.

For the sandwich system, an additional collagen gel solution will bedistributed over the cells after 1 day of culture. The culture mediumwill be carefully removed to ensure that the second layer of collagengel is evenly spread over the entire plate. The culture plates will beincubated at 37° C. to allow gelation and attachment of the second gellayer before the medium was replaced. The culture medium will be changeddaily. Medium samples will be stored at 20° C. for further analysis.

Hepatocytes cultured between layers of gelled collagen maintain athree-dimensional cuboidal shape and distribution of cytoskeletalproteins similar to that observed in vivo.

Optimization of Bile Canalicular Network Formation

To optimize taurocholate accumulation and biliary excretion, particularculture medium, such as Williams' medium E and Dulbecco's modifiedEagle's medium can be used in the sandwich hepatocyte culture.

Test Articles

The FXR agonist intended for study is obeticholic acid, also known as“OCA” and 6-ethyl chenodeoxycholic acid (6-ECDCA).

PPAR-alpha agonists intended for study include one or more ofclofibrate, gemfibrozil, ciprofibrate, bezafibrate, and fenofibrate).

A dual PPAR-alpha/delta agonist is 2-[2,6dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxyl]-2-methylpropanoicacid.

A PPARS (delta) agonist intended for study is GW501516.

Statins (HMG-CoA reductase inhibitors) intended for study includeatorvastatin (Lipitor), rosuvastatin (Crestor) and simvastatin (Zocor).

Example 5: Evaluate Effects of Individual Administration of TestArticles on Lipid Profiles

The potential of 5 test articles, an FXR agonist, a PPAR-alpha agonist,a PPAR-delta agonist, a dual PPAR-alpha/delta agonist (or alternatively,PPAR-alpha agonist and PPAR-delta agonist together), and a statin willbe assessed to determine the ability to alter cholesterol synthesis andthe lipid profile in human hepatocytes. Changes will be evaluated insandwich-cultured human hepatocytes (SCHH) following 72 hours ofexposure to test articles at 3 different concentrations. Dosingsolutions will be made fresh daily in culture media and dosing of SCHHwill occur daily for 3 days. The experiment will be performed in 24-wellformat using one (1) lot of Transporter Certified™ Human Hepatocytes(N=1). Each test condition will be performed in three (3) wells toprovide triplicate data (expressed as mean±standard deviation). Solventcontrol treated plates will be used as a control and evaluated forbaseline function. At the end of the test period, internal standard willbe added to individual wells, followed by addition of the extractionreagent for global lipid profiling. The samples will be shaken for 1hour at room temperature, and centrifuged. The supernatant will beevaporated to dryness under nitrogen, re-suspended and analyzed.

Global lipid profiling will be performed using Ultra-Performance LiquidChromatography (UPLC) and high-resolution MS. Methyl-t-butyl sampleextracts will be analyzed on UPLC-MS (Synapt G2 Ion-Mobility QToF)instrumentation, in ESI+ and ESI-mode, to cover a wide range of lipidpolarity and chemical composition. Initially UPLC will be applied toevaluate compound effects on a large array (5000 to 8000) of lipids,including multiple esters of cholesterol. Colorimetric analysis will beperformed to measure total cholesterol. Based on abundance most will beglycerophospholipids, however, a large number of classes will beevaluated. Profiles will be evaluated to identify potential effects onindividual lipids. Identification of specific lipids can be performedagainst standards using assay retention time, accurate mass andfragmentation. Depending on the outcome, specific lipids or lipidclasses may be identified for evaluation in Examples 6 and 7. Aconfirmatory study will be repeated in two additional lots ofTransporter Certified™ Human Hepatocytes (N=2).

Example 6: Evaluate Effects of Dual Combinations of Test Articles onLipid Profiles

Combinations of FXR agonist, with each of a PPAR-alpha agonist, aPPAR-delta agonist, a PPAR-alpha and delta dual agonist (or, in thealternative, an FXR agonist with a PPAR alpha agonist and a PPAR deltaagonist), and/or a statin will be evaluated for their potential to altercholesterol synthesis and the lipid profile in human hepatocytes.Specific combinations evaluated will be:

-   -   FXR agonist with PPAR-alpha agonist    -   FXR agonist with PPAR-delta agonist    -   FXR agonist with PPAR-alpha and delta dual agonist and/or FXR        agonist with PPAR-alpha agonist and PPAR-delta agonist    -   FXR agonist with a statin

Changes will be evaluated in sandwich-cultured human hepatocytes (SCHH)following 72 hours of exposure to test articles at 3 differentconcentrations. Dosing solutions will be made fresh daily in culturemedia and dosing of SCHH will occur daily for 3 days. The experimentwill be performed in 24well format using one (1) lot of TransporterCertified™ Human Hepatocytes (N=1). Each test condition will beperformed in three (3) wells to provide triplicate data (expressed asmean±standard deviation). Samples will be prepared and analyzed forglobal lipid profiling as detailed in Example 5. Alterations in lipidprofiles and cholesterol synthesis will be compared with effects fromindividual administration in Example 2.

Example 7: Evaluate Effects of Triple Combinations of Test Articles onLipid Profiles

The triple combination of an FXR agonist, PPAR-alpha agonist, PPAR-deltaagonist, PPAR-alpha and delta dual agonist (or PPAR-alpha agonist incombination with PPAR-delta agonist), and/or a statin will be evaluatedfor the potential to alter cholesterol synthesis and the lipid profilein human hepatocytes. Changes will be evaluated in sandwich-culturedhuman hepatocytes (SCHH) following 72 hours of exposure to test articlesat 3 different concentrations. Dosing solutions will be made fresh dailyin culture media and dosing of SCHH will occur daily for 3 days. Theexperiment will be performed in 24-well format using one (1) lot ofTransporter Certified™ Human Hepatocytes (N=1). Each test condition willbe performed in three (3) wells to provide triplicate data (expressed asmean±standard deviation). Samples will be prepared and analyzed forglobal lipid profiling as detailed in Example 2. Alterations in lipidprofiles and cholesterol synthesis will be compared with effects fromcombinations administered in Example 2. Specific combinations evaluatedwill be:

-   -   FXR agonist with a PPAR-alpha agonist, and a statin    -   FXR agonist with a PPAR-delta agonist, and a statin    -   FXR agonist with a PPAR-alpha and delta dual agonist (or, in the        alternative, a PPAR-alpha agonist and a PPAR-delta agonist), and        a statin        Samples will be prepared and analyzed for global lipid profiling        as detailed in Example 4. Alterations in lipid profiles and        cholesterol synthesis will be compared with effects from        combinations administered in Examples 4 and 5.

Example 8: Animal Studies Animals

Animals will be housed individually in standard cages at 22° C. in a12:12-h light-dark cycle. Male C57BL/6 mice (Jackson Laboratories, BarHarbor, Me.) will be allowed ad libitum access to a diet enriched in fat(40% kcal, Primex partially hydrogenated vegetable oil shortening),fructose (22% by wt), and cholesterol (2% by wt) (Research Diets, NewBrunswick, N.J., cat. no. D09100301). A low-fat diet (10% kcal;hereafter referred to as LFD) with no fructose or cholesterol will beused as a control diet (Research Diets, cat. no. D09100304). The use ofthis validated LFD establishes a group of control mice that maintain a“normal” hepatic phenotype for comparison with animals fed theexperimental diet.

Treatment Groups

-   -   Control HFD:    -   Control LFD:    -   HFD+FXR agonist:    -   HFD+PPARα (i.e., fenofibrate, gemfibrozil, bezofibrate, or        ciprofibrate):    -   HFD+PPARδ (i.e., GW501516):    -   HFD+PPARα+PPARδ:    -   HFD+dual PPARα/δ (i.e., GFT505):    -   HFD+statin (i.e., atorvastatin, simvastatin, rosuvastatin)    -   HFD+FXR agonist+PPARα:    -   HFD+FXR agonist+PPARδ:    -   HFD+FXR agonist+PPARα+PPARδ:    -   HFD+FXR agonist+dual PPARα/δ:    -   HFD+FXR agonist+statin:

Histology and Digital Image Analysis

At termination, right medial and/or left lateral lobes of the liver(>50% of each lobe harvested) will be excised and fixed in 10%neutral-buffered formalin (at least 7 days at room temperature). Theliver tissue will be carefully excised to select similarly sizedsections representative of both the tissue edge and center. Liver tissuewill be paraffin embedded, sectioned (5 μm), and mounted. Hematoxylinand eosin stains will be used for morphological analyses, and Masson'strichrome and Sirius red stains will be used for assessment of hepaticfibrosis. Histopathological analysis will be performed by a pathologistblinded to the study. NAFLD and NASH will be scored by use of criteriaoutlined by Kleiner and colleagues. For quantitative assessment offibrosis, whole Sirius red-stained sections will be scanned by use ofthe ScanScope CS whole slide scanning system (Aperio, Vista, Calif.) at×20 magnification. Images will be extracted and Sirius red-stainedcollagen profiles from entire tissues will be measured by the colorcube-based method with Image-Pro Analyzer software (MediaCyberneticsv.6.2, Bethesda, Md.). Total collagen staining (reported as % of totalarea) will be assessed from three to four representative sections fromeach animal (except for the comprehensive liver fibrosis assessmentexperiment where additional sections will be evaluated). Allhistological analyses will be performed blinded.

Liver Biopsy

Mice will be anesthetized with isoflurane (2-3%) in 100% oxygen. A smallabdominal incision, ˜0.5 cm left of midline will be made and the leftlateral lobe of the liver will be exposed. A wedge of liver tissue (˜50mg) will be excised from the distal portion of the lobe, immediatelyplaced in a vial, and snap frozen in liquid nitrogen. A wedge ofabsorbable gelatin sponge (GelFoam, Pfizer, NY) will be inserted intothe cut edges of the liver. Once hemostasis is achieved (typicallywithin 1 min) and the gelatin sponge well-adhered to the biopsy site,the liver will be returned to the abdominal cavity, the abdominal wallsutured, and the skin stapled. Mice will receive a single injection ofbuprenorphine (0.05 mg/kg, subcutaneous) at the time of the surgery tocontrol postoperative pain. Sham operated mice will undergo an identicalprocedure except no incision made in the liver.

Plasma and Serum Analysis

Plasma glucose, triglycerides, total cholesterol, alanineaminotransferase (ALT), and aspartate aminotransferase (AST) levels willbe measured by using an Olympus AU400e Bioanalyzer (Olympus AmericaDiagnostics, Center Valley, Pa.). Plasma samples will be diluted 1:10with PBS for measurement of ALT and AST. Total plasma adiponectin andfasting serum insulin will be measured according to the manufacturer'sinstructions with commercially available electrochemiluminescence kits(Meso Scale Discovery, Gaithersburg, Md.).

Quantification of total hepatic lipid and collagen content Total hepaticlipid will be extracted from the liver using a protocol adapted fromFolch et al. Frozen liver tissue (˜0.3 g) will be homogenized in 10 mlof 2:1 chloroform-methanol solution. The homogenate will be filteredusing fat-free filter paper and funneled into a preweighed 15-ml glassvial. An additional 5 ml of 2:1 chloroform-methanol will be addedfollowed by 2.5 ml of 0.9% NaCl. The lipids will be separated bycentrifugation at 1,800 g, 10° C. for 5 min, the aqueous layer will bediscarded, and the tube will be flushed with nitrogen until the lipidpellet will be dry. The tube containing the lipid pellet will bereweighed, and total lipid extracted per gram of total liver will becalculated. Total collagen content in the liver will be measured bycolorimetric determination of hydroxyproline residues by acid hydrolysisof collagen (Quickzyme, Leiden, Netherlands).

Determination of Extractable Collagen-1α1 Protein by Protein Blot

Tissue cores (50-100 mg) will be collected from the left lateral lobe ofthe liver, snap frozen in liquid nitrogen, and stored at −80° C. untilprocessed. The tissue will be homogenized in lysis buffer containingprotease inhibitors. Protein concentration of the cleared supernatantwill be measured with a BCA protein assay kit (Pierce, Rockford, Ill.).Liver tissue lysates (˜50 μg) will be separated on reducing 4-12% Nupagegels (Life Technologies, Carlsbad, Calif.) and transferred tonitrocellulose membranes. Membranes will be cut between the 50- and60-kDa markers and blocked with 5% Blotto. The upper half will be probedwith anti-collagen-1% 1 (1:1,000; cat. no. NBP1-30054; NovusBiologicals, Littleton, Colo.), which detects the COOH-terminaltelopeptide portion of the collagen-1%1 protein. For normalization, thelower half will be probed with anti-glyceraldehyde 3-phosphatedehydrogenase (GAPDH, 1:7,500; cat. no. 3683; Cell SignalingTechnologies, Danvers, Mass.). Following incubation with horseradishperoxidase anti-rabbit antibody, protein expression will be detectedwith enhanced chemiluminescence (Thermo Scientific, Rockford, Ill.), anddensitometry will be performed with a FluorChem System (CellBiosciences, Santa Clara, Calif.). Densitometry analysis of collagen-1α1will include both the 140-kDa mature protein as well as a slightlylarger band, corresponding to a glycosylated form or a partiallyprocessed collagen-1α1 protein.

Hepatic Gene Expression Changes

Tissue samples from the left lateral lobe of the liver will be harvestedwith a 6-mm tissue coring tool or by the biopsy method, snap frozen inliquid nitrogen, and stored at −80° C. until processed. Total RNA fromliver samples (˜50-150 mg) will be extracted by use of TRI Reagent (LifeTechnologies) and then further purified with a Qiagen RNeasy Plus Minikit (Qiagen, Valencia, Calif.). RNA integrity will be determined byusing the Agilent 6000 nano kit on a Bioanalyzer 2100 (AgilentTechnologies, Santa Clara, Calif.). cDNA will be prepared by using theHigh Capacity cDNA Reverse Transcription Kit (Life Technologies).Changes in gene expression will be confirmed by TaqMan gene expressionassays-on-demand and Universal Master Mix (Life Technologies) on an ABIPrism 7900HT instrument (Applied Biosystems, Foster City, Calif.).Change in gene expression will be calculated by the comparativethreshold cycle (CT) method with peptidylprolyl isomerase A (Ppia) andGapdh for normalization. For gene arrays, cDNA samples will be run onMouse Fibrosis RT2 Profiler PCR Arrays (PAMM-120C, RT2 SYBR Green/ROXqPCR Master Mix; SABiosciences) by using the ABI Prism 7900HT FastReal-Time PCR System (Applied Biosystems). Changes in gene expression onthe array will be calculated by the comparative CT method usingDataAssist v3.0 software (Applied Biosystems/Life Technologies). Amongthe five housekeeping genes included in the Mouse Fibrosis RT2 ProfilerPCR Array, hypoxanthine phosphoribosyltransferase 1 (Hprt) and Gapdhhave the most stable expression according to the stability scorescalculated by DataAssist v3.0 software. The mean of the chosenendogenous control genes will be used as the normalization factor tocalculate the relative expression of each gene. To confirm the resultsobtained by using the fibrosis array, TaqMan Gene expression assays willbe conducted for a selection of genes determined by the array to beupregulated, downregulated, or unchanged.

Example 9: Clinical Trial

A multicenter, double-blind, placebo-controlled, parallel group,randomized clinical trial was conducted in patients with non-cirrhotic,non-alcoholic steatohepatitis to assess treatment with obeticholic acidgiven orally (25 mg daily) or placebo for 72 weeks. Patients wererandomly assigned 1:1 using a computer-generated, centrally administeredprocedure, stratified by clinical center and diabetes status. Theprimary outcome measure was improvement in centrally scored liverhistology defined as a decrease in non-alcoholic fatty liver diseaseactivity score by at least 2 points without worsening of fibrosis frombaseline to the end of treatment. Change in alanine aminotransferase at24 weeks was measured: relative change in alanine aminotransferase −24%,95% CI −45 to −3.

Study Design and Participants

Patients were enrolled in the study according to the following inclusioncriteria: 18 years or older at the time of screening, histologicalevidence of definite or borderline non-alcoholic steatohepatitis basedupon a liver biopsy obtained 90 days or less before randomization, and ahistological non-alcoholic fatty liver disease (NAFLD) activity score of4 or more with a score of 1 or more in each component of the score(steatosis scored 0-3, ballooning 0-2, and lobular inflammation 0-3).Grading and staging of biopsies for the purposes of enrollment werecarried out at the site of enrollment. Exclusion criteria include thepresence of cirrhosis, other causes of liver disease, substantialalcohol consumption (>20 g/day for women or >30 g/day for men), or otherconfounding conditions (see below).

Randomization and Masking

Patients meeting eligibility criteria were randomly assigned (1:1) tooral obeticholic acid, 25 mg once-daily, or placebo. Obeticholic acidand placebo were provided as identical tablets in identical containerslabelled with code numbers. Patients, investigators, clinical sitestaff, and pathologists will be masked to treatment assignment.

Procedures

After randomization, patients returned for study visits at weeks 2, 4,and 12, and then every 12 weeks until completion of treatment at week72, and then 24 weeks later. Blood samples were obtained at these visitsfor routine biochemical tests and assessment of fasting concentrationsof lipids, glucose, and insulin. Body weight, height, and waist and hipcircumferences were measured at the initial assessment and designatedinterim times. All patients received standard recommendations on healthyeating habits, weight reduction, exercise, and the management ofhypertension, hypercholesterolemia, and diabetes when indicated.

Baseline and end-of-treatment liver biopsies were centrally assessed asa group for consensus scoring of each component of the NAFLD activityscore, determined fibrosis stage, and assigned a diagnosis ofnon-alcoholic steatohepatitis, borderline non-alcoholic steatohepatitis,or not non-alcoholic steatohepatitis.

Inclusion and Exclusion Criteria

Patients who meet any of the following exclusion criteria wereconsidered ineligible for enrollment: 1) Current or history ofsignificant alcohol consumption for a period of more than 3 consecutivemonths within 1 year prior to screening (significant alcohol consumptionwas defined as more than 20 g/day in females and more than 30 g/day inmales, on average); 2) Inability to reliably quantify alcoholconsumption based upon site investigator's judgment; 3) Use of drugshistorically associated with NAFLD for more than 2 weeks in the yearprior to randomization; 4) Prior or planned bariatric surgery orprocedure; 5) Uncontrolled diabetes defined as HbA1c of 80.3 mmol/mol orhigher within 60 days prior to enrollment; 6) Presence of cirrhosis onliver biopsy, 7) Platelet count <100×109/L; 8) Clinical evidence ofhepatic decompensation as defined by the presence of any of thefollowing abnormalities: serum albumin less than 32 g/L, INR greaterthan 1.3, direct bilirubin greater than 22.2 μmol/L, or a history ofesophageal varices, ascites, or hepatic encephalopathy; 9) Evidence ofother forms of chronic liver disease: hepatitis B as defined by presenceof hepatitis B surface antigen (HBsAg), hepatitis C as defined bypresence of hepatitis C virus (HCV) RNA or positive hepatitis C antibody(anti-HCV), evidence of ongoing autoimmune liver disease as defined bycompatible liver histology, primary biliary cirrhosis as defined by thepresence of at least 2 criteria (biochemical evidence of cholestasisbased mainly on alkaline phosphatase elevation, presence ofanti-mitochondrial antibody [AMA], and histologic evidence ofnonsuppurative destructive cholangitis and destruction of interlobularbile ducts), primary sclerosing cholangitis, Wilson's disease as definedby ceruloplasmin below the limits of normal and compatible liverhistology, alpha-1-antitrypsin (A1AT) deficiency as defined bydiagnostic features in liver histology (confirmed by alpha-1 antitrypsinlevel less than normal, exclusion at the discretion of the siteinvestigator), history of hemochromatosis or iron overload as defined bypresence of 3+ or 4+ stainable iron on liver biopsy, drug-induced liverdisease as defined on the basis of typical exposure and history, knownbile duct obstruction, suspected or proven liver cancer, or any othertype of liver disease other than NASH; 10) Serum alanineaminotransferase (ALT) greater than 300 U/L; 11) Serum creatinine of176.8 μmol/L or greater; 12) Inability to safely obtain a liver biopsy;13) History of biliary diversion; 14) Known positivity for HumanImmunodeficiency Virus (HIV) infection; 15) Active, serious medicaldisease with likely life expectancy less than 5 years; 16) Activesubstance abuse including inhaled or injection drugs in the year priorto screening; 17) Pregnancy, planned pregnancy, potential for pregnancyand unwillingness to use effective birth control during the trial, orbreast feeding; 18) Participation in an IND trial in the 30 days beforerandomization; 19) Any other condition which, in the opinion of the siteinvestigator, would impede compliance or hinder completion of the study;or 20) Failure to give informed consent.

Statistical Analysis

The primary outcome and binary secondary outcomes were analyzed usingthe Mantel-Haenszel test; continuous secondary outcomes were analyzedusing ANCOVA models relating change in the continuous outcome frombaseline to 72 weeks to treatment group and to the baseline value of theoutcome. Statistical analyses were performed with SAS (SAS Institute2011, Base SAS 9.3 Procedures Guide) and Stata (StataCorp 2013, StataStatistical Software: release 13).

Outcomes

The primary outcome measure was improvement in centrally scored liverhistology defined as a decrease in NAFLD activity score by at least 2points without worsening of fibrosis from baseline to the end oftreatment. Worsening of fibrosis was defined as any numerical increasein the stage. Secondary histological outcomes include resolution ofnon-alcoholic steatohepatitis, change in NAFLD activity score, andchanges in the individual scores for hepatocellular ballooning,steatosis, lobular and portal inflammation, and fibrosis. Improvement infibrosis was defined as any numerical decrease in the stage. Fibrosisstages 1a, 1b, and 1c were considered stage 1 for the purposes ofanalysis. Other secondary outcomes include changes from baseline to 72weeks in serum aminotransferase and γ-glutamyl transpeptidaseconcentrations, fasting homoeostasis model of assessment of insulinresistance (HOMA-IR), anthropometric measures (weight, bodymass index,waist-to-hip ratio, waist circumference), and health-relatedquality-of-life scores.

Example 10: Data Analysis

A subanalysis of the data obtained in Example 9 was performed to assessthe effect of statins on low density lipoprotein cholesterol (LDL-C)levels. The aims of these secondary analyses were to determine theeffect of OCA versus placebo in the subgroup of patients with moresevere NASH and to assess the effects of concomitant statin use on serumLDL cholesterol.

Subject data were assessed in three groups as follows:

-   -   Group A (n=64, no statin) included subjects in the obeticholic        acid (OCA) treatment arm who were not on a statin at baseline        (Day 0) and who do not initiate a statin throughout the course        of the study up to and including Week 72.    -   Group B (n=47, baseline statin) included subjects in the OCA        treatment arm who were on a statin at baseline and who continued        on the statin during the study up to and including Week 72.    -   Group C (n=23, new statin) included subjects in the OCA        treatment arm who were not on a statin at baseline but initiated        statin treatment at a time after baseline up to and including        Week 72.        -   The following calculations were performed for OCA treated            subjects in Groups A, B, and C.    -   Mean and median characteristics listed below will be evaluated        at baseline and at Week 72.        -   Laboratory values: LDL-C, high density lipoprotein            cholesterol (HDL-C), alanine and aspartate aminotransferase,            gamma glutamyl transferase        -   Age        -   Gender: percentage male, percentage female        -   Percentage diabetic        -   Histology: steatosis, ballooning, inflammation, fibrosis    -   Mean and median percentage change at Week 72 from baseline of        the above characteristics will be evaluated.

Results

LDL cholesterol increased during OCA treatment in patients on statins atbaseline, but levels did not exceed those of Placebo-treated patientsnot on statins. Statin initiation during OCA treatment reversed LDL tobelow pre-OCA baseline levels. As shown in FIG. 8, the OCA-related LDLincrease appeared to be reversed by initiating statin therapy during OCAtreatment.

1. (canceled)
 2. A method of treating or preventing an FXR mediateddisease or condition or a condition related to elevated lipid levels ina subject in need thereof, comprising administering to the subject acompound of formula (1):

or a pharmaceutically acceptable salt or amino acid conjugate thereof,and a fibrate.
 3. The method of claim 2, wherein the FXR mediateddisease or condition is selected from primary biliary cirrhosis (PBC),primary sclerosing cholangitis (PSC), portal hypertension, bile aciddiarrhea, a chronic liver disease, nonalcoholic fatty liver disease(NAFLD), nonalcoholic steatohepatitis (NASH), hepatitis C infection, analcoholic liver disease, liver damage due to progressive fibrosis, liverfibrosis, a cardiovascular disease, and hyperlipidemia.
 4. The method ofclaim 2, wherein the FXR mediated disease or condition is a cholestaticliver disease selected from primary biliary cirrhosis (PBC), primarysclerosing cholangitis (PSC), drug-induced cholestasis, hereditarycholestasis, biliary atresia, and intrahepatic cholestasis of pregnancy.5. The method of claim 4, wherein the cholestatic liver disease is PBC.6. The method of claim 3, wherein the FXR mediated disease or conditionis NASH.
 7. The method of claim 3, wherein the FXR mediated disease orcondition is liver fibrosis.
 8. The method of claim 2, wherein thecompound of formula (1) is in an amount of 1-30 mg.
 9. The method ofclaim 2, wherein the compound of formula (1) is in an amount of 5-25 mg.10. The method of claim 2, wherein the fibrate is in an amount of 80-400mg.
 11. The method of claim 2, wherein the fibrate is in an amount of100-300 mg.
 12. The method of claim 2, wherein the fibrate is in anamount of 100 mg.
 13. The method of claim 2, wherein the fibrate is inan amount of 200 mg.
 14. The method of claim 2, wherein the fibrate isin an amount of 400 mg.
 15. The method of claim 2, wherein the fibrateis fenofibrate, gemfibrozil, bezafibrate, or ciprofibrate.
 16. A methodof treating or preventing an FXR mediated disease or condition or acondition related to elevated lipid levels in a subject in need thereof,comprising administering to the subject a pharmaceutical compositioncomprising a combination of therapeutic agents consisting of a compoundof formula (1):

or a pharmaceutically acceptable salt or amino acid conjugate thereof,and a fibrate; wherein the pharmaceutical composition further optionallycomprises one or more pharmaceutically acceptable carriers.
 17. Themethod of claim 16, wherein the FXR mediated disease or condition isselected from primary biliary cirrhosis (PBC), primary sclerosingcholangitis (PSC), portal hypertension, bile acid diarrhea, a chronicliver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), hepatitis C infection, an alcoholic liverdisease, liver damage due to progressive fibrosis, liver fibrosis, acardiovascular disease, and hyperlipidemia.
 18. The method of claim 16,wherein the FXR mediated disease or condition is a cholestatic liverdisease selected from primary biliary cirrhosis (PBC), primarysclerosing cholangitis (PSC), drug-induced cholestasis, hereditarycholestasis, biliary atresia, and intrahepatic cholestasis of pregnancy.19. The method of claim 18, wherein the cholestatic liver disease isPBC.
 20. The method of claim 17, wherein the FXR mediated disease orcondition is NASH.
 21. The method of claim 17, wherein the FXR mediateddisease or condition is liver fibrosis.
 22. The method of claim 16,wherein the compound of formula (1) is in an amount of 1-30 mg.
 23. Themethod of claim 16, wherein the compound of formula (1) is in an amountof 5-25 mg.
 24. The method of claim 16, wherein the fibrate is in anamount of 80-400 mg.
 25. The method of claim 16, wherein the fibrate isin an amount of 100-300 mg.
 26. The method of claim 16, wherein thefibrate is in an amount of 100 mg.
 27. The method of claim 16, whereinthe fibrate is in an amount of 200 mg.
 28. The method of claim 16,wherein the fibrate is in an amount of 400 mg.
 29. The method of claim16, wherein the fibrate is fenofibrate, gemfibrozil, bezafibrate, orciprofibrate.
 30. A method of treating or preventing an FXR-mediateddisease or condition or a condition related to elevated lipid levels ina subject in need thereof, comprising administering to the subject apharmaceutical composition comprising a combination of therapeuticagents consisting of a compound of formula (1):

or a pharmaceutically acceptable salt or amino acid conjugate thereof,and bezafibrate; wherein the pharmaceutical composition furtheroptionally comprises one or more pharmaceutically acceptable carriers.31. The method of claim 30, wherein the FXR mediated disease orcondition is selected from primary biliary cirrhosis (PBC), primarysclerosing cholangitis (PSC), portal hypertension, bile acid diarrhea, achronic liver disease, nonalcoholic fatty liver disease (NAFLD),nonalcoholic steatohepatitis (NASH), hepatitis C infection, an alcoholicliver disease, liver damage due to progressive fibrosis, liver fibrosis,a cardiovascular disease, and hyperlipidemia.
 32. The method of claim30, wherein the FXR mediated disease or condition is a cholestatic liverdisease selected from primary biliary cirrhosis (PBC), primarysclerosing cholangitis (PSC), drug-induced cholestasis, hereditarycholestasis, biliary atresia, and intrahepatic cholestasis of pregnancy.33. The method of claim 32, wherein the cholestatic liver disease isPBC.
 34. The method of claim 31, wherein the FXR mediated disease orcondition is NASH.
 35. The method of claim 31, wherein the FXR mediateddisease or condition is liver fibrosis.
 36. The method of claim 30,wherein the compound of formula (1) is in an amount of 1-30 mg.
 37. Themethod of claim 30, wherein the compound of formula (1) is in an amountof 5-25 mg.
 38. The method of claim 30, wherein bezafibrate is in anamount of 80-400 mg.
 39. The method of claim 30, wherein bezafibrate isin an amount of 100-300 mg.
 40. The method of claim 30, whereinbezafibrate is in an amount of 100 mg.
 41. The method of claim 30,wherein bezafibrate is in an amount of 200 mg.
 42. The method of claim30, wherein bezafibrate is in an amount of 400 mg.
 43. The method ofclaim 30, wherein the composition is a single unit dosage form.
 44. Themethod of claim 43, wherein the single unit dosage form is a tablet orcapsule.
 45. The method of claim 44, wherein the compound of formula (1)is in an amount of 5-25 mg and bezafibrate is in an amount of 100-300mg.
 46. The method of claim 44, wherein the compound of formula (1) isin an amount of 4-26 mg and bezafibrate is in an amount of 100-300 mg.